Ion-selective quantum dots

ABSTRACT

The invention provides ion-selective sensors comprising quantum dots capable of selectively measuring ions, e.g., Na + , K + , Cl − , etc., in various environments, including in the cytosol of a living cell. Quantum dots are attractive probes for microscopy due to their photophysical advantages over fluorescent dyes, including prolonged photostability, brightness and quantum efficiency. In certain embodiments, a sensor comprises one or more quantum dots, a pH-sensitive dye, and optionally an ion-selective component such as an ionophore. These elements may, for example, be disposed in a polymer matrix. In certain embodiments, the sensors may detect ionic analytes by selective ion extraction by the polymer, thereby inducing a pH change within the sensor which in turn changes the absorbance of the pH-sensitive dye. The change of absorbance may in turn attenuate the intensity of detectable emissions, e.g., fluorescence, from the quantum dot by directly absorbing its fluorescence emission.

CROSS-REFERENCE TO RELATED CASES

This application claims the benefit of U.S. Provisional PatentApplication No. 60/834,973, entitled “Ion-Selective Quantum Dots,” filedon Jul. 31, 2006, the entire disclosure of which is hereby incorporatedby reference as if set forth herein in its entirety.

BACKGROUND OF THE INVENTION

Ion channels lie at the heart of the dynamics of excitable cells andcontrol the action potential responsible for the beat of a cardiac cellor the firing of a synapse. Traditionally, calcium has been the moststudied ion in the intracellular space, and as a result a good deal isunderstood about calcium ion dynamics, regulation and impact on thecell. While it is generally believed that sodium is essential to thefunction of excitable cells, its role is not as well understood. Sodiumchannels occur at sites of action potential generation in neurons andare responsible for the upstroke of the action potential in cardiaccells. Sodium channelopathies are known to be responsible for majorclasses of disease, including epilepsy and hypertension, as well asleading to potentially fatal arrhythmias in Long QT syndrome. Inaddition, calcium dynamics are often dependent upon sodium, through thesodium/calcium exchanger. The exchanger is responsible for the rapidexport of calcium from the cell and is driven by the transmembranesodium gradient. While channel labeling studies have elucidated thelocation of sodium channels, little is known about the function,dynamics and impact of sodium channels.

There is a wide selection of methods for measuring ion flux in cellsincluding, for example, patch clamping, fluorescent indicator dyes,polymer-based ion-selective nanosensors and dye-loaded liposomes. Thereare, however, disadvantages to the available methods of ion monitoring.For example, whole-cell patch clamp monitoring which involves contactingcells with a pulled glass capillary to electrically monitor ionchannels, can be performed on single cells but not reliably in ahigh-throughput fashion. Fluorescent ion-indicator dyes while effectivefor monitoring certain ions, lack selectivity for other physiologicalions and are easily photobleached. Ion-selective nanosensors (e.g.,probes encapsulated by biologically localized embedding (PEBBLEs)) whichcomprise a fluorescent indicator dye display fast response time to ionsas they diffuse into the polymer matrix, however, the components of thenanosensors, such as the fluorescent dye, are also prone tophotobleaching. Dye-loaded liposomes are highly biocompatible due totheir lipid construction but are limited in their range of detectableanalytes particularly to gases.

New methods are needed for monitoring ion fluctuations in cells that areselective for a particular ionic analyte such as sodium, arebiocompatible and have prolonged sensor lifespans. Further, sensors thatare small enough to enter the cell but emit a strong enough signal to bemeasured extracellularly would be highly desired.

SUMMARY OF THE INVENTION

The invention provides ion-selective sensors comprising quantum dotscapable of selectively measuring ions, e.g., Na⁺, K⁺, Cl⁻, etc., in thecytosol of a single living cell. Quantum dots are attractive probes formicroscopy due to their photophysical advantages over fluorescent dyes,including prolonged photostability, brightness and quantum efficiency.In certain embodiments, a sensor comprises one or more quantum dots, apH-sensitive dye, and optionally an ion-selective component such as anionophore. These elements may, for example, be disposed in a polymermatrix. In certain embodiments, the sensors may detect ionic analytes byselective ion extraction by the polymer, thereby inducing a pH changewithin the sensor which in turn changes the absorbance of thepH-sensitive dye. The change of absorbance may in turn attenuate theintensity of detectable emissions, e.g., fluorescence, from the quantumdot by directly absorbing its fluorescence emission.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of a cell assay system according to anillustrative embodiment of the invention.

FIG. 2 is a representation of two exemplary modes of operation of thequantum dot incorporated sensor for the detection of cationic analytes.In Mode A, the sensor fluoresces in the presence of the ionic analyte,In Mode B, the sensor fluoresces in the absence of ionic analyte.

FIG. 3 is a representation of two exemplary modes of operation of thequantum dot incorporated sensor for the detection of anionic analytes.In Mode A, the sensor fluoresces in the presence of the ionic analyte,In Mode B, the sensor fluoresces in the absence of ionic analyte.

FIG. 4 is a is a representation of a sensor coated with a surfacemodifier such as PEG.

FIGS. 5 a-d are cross sections of various optical sensor arrangementssuitable for use in various implementations of a cell assay system.

FIGS. 6 a-b presents an embodiment of a microfluidic device in accordwith the present invention.

FIG. 7 shows the selectivity of the nanosensor of the invention for iondetection.

FIG. 8 depicts the experimental response to sodium. a) Spectral responseof immobilized sensors to increasing concentrations of sodium. b)Calibration curve of ratiometric sensors.

FIG. 9 depicts the spectral overlap of a quantum dot that fluoresces at655 nm and the absorbance of a chromoionophore at varying sodiumconcentrations.

FIG. 10 depicts biocompatibility of nanosensors in HEK cells. HEK cellswere incubated with either control (water), nanosensors without quantumdots (nans), quantum dot nanosensors, 100 nm gold nanoparticles, or 20nm latex beads (a negative control).

FIG. 11 is a confocal image of nanosensors without quantum dots loadedinto an HEK 293 cell.

FIG. 12 shows a LIVE/DEAD assay wherein nanosensors with quantum dotswere loaded into HEK 293 cells overnight and then stained. The greenindicates healthy cells, while the red stains the nuclei of dead cells.No difference in the ratio of live to dead cells was noted betweennanosensor loaded cells and control (no nanosensors).

FIG. 13 depicts a. fluorescence image of an isolated neonatal ratventricular myocyte loaded with sodium-selective nanosensors. b. thefluorescence collected from a nanosensor in a cardiac cell duringstimulation

DETAILED DESCRIPTION OF THE INVENTION

In brief overview, embodiments of the present invention provide systems,methods, and devices for measuring ionic analytes. In exemplaryembodiments, sensors are placed inside or outside a cell. Changes inemissions from the sensor indicate the ion concentrations and fluxesfrom the cell. In certain aspects, the sensors comprise a polymer, afluorescent semiconductor nanocrystal (also known as a quantum dot™particle) that fluoresces at a first wavelength, and a chromoionophorethat absorbs photons of the first wavelength in one state and does notabsorb photons of the first wavelength in a second state. In monitoringionic analytes, the chromoionophore changes state in response to protonconcentration (i.e., the protonated chromoionophore is one state whilethe deprotonated chromoionophore is a second state). To monitor aspecific analyte, an ionophore that selectively associates with specificions or groups of ions is included in the sensor. Once the ionophoreassociates with a cationic analyte (e.g., Na⁺ associates with aNa⁺-selective ionophore), for example, protons are displaced from thesensor to equilibrate charge, altering the state of the chromoionophore.The fluorescence emitted from the sensor indicates the state of thechromoionophore which correlates to the presence and/or concentration ofthe ionic analyte. Sensors that use fluorescent dyes instead of quantumdots are disclosed in U.S. patent application Ser. No. 11/522,169, filedSep. 15, 2006, the disclosure of which is incorporated herein byreference.

In certain embodiments, the sensor includes an ionophore, achromoionophore, a quantum dot, and optionally one or more additives.The components are typically embedded in a polymer. In certainembodiments, the polymer comprises poly(caprolactone) (PCL), ethylenevinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lacticacid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolicacid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA),poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, silicones, polyalkylenes such aspolyethylene, polypropylene, and polytetrafluoroethylene, polyalkyleneglycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO),polyalkylene terephthalates such as poly(ethylene terephthalate),polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such aspoly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride)(PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS),polyurethanes, derivatized celluloses such as alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers ofacrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) (jointlyreferred to herein as “polyacrylic acids”), and copolymers and mixturesthereof, polydioxanone and its copolymers, polyhydroxyalkanoates,poly(propylene fumarate), polyoxymethylene, poloxamers,poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), trimethylene carbonate,polyvinylpyrrolidone, and the polymers described in Shieh et al., 1994,J. Biomed. Mater. Res., 28, 1465-1475, and in U.S. Pat. No. 4,757,128,Hubbell et al., U.S. Pat. Nos. 5,654,381; 5,627,233; 5,628,863;5,567,440; and 5,567,435. Other suitable polymers includepolyorthoesters (e.g. as disclosed in Heller et al., 2000, Eur. J.Pharm. Biopharn., 50:121-128), polyphosphazenes (e.g. as disclosed inVandorpe et al., 1997, Biomaterials, 18:1147-1152), andpolyphosphoesters (e.g. as disclosed in Encyclopedia of Controlled DrugDelivery, pp. 45-60, Ed. E. Mathiowitz, John Wiley & Sons, Inc. NewYork, 1999), as well as blends and/or block copolymers of two or moresuch polymers. The carboxyl termini of lactide- and glycolide-containingpolymers may optionally be capped, e.g., by esterification, and thehydroxyl termini may optionally be capped, e.g., by etherification oresterification. In certain embodiments, the polymer comprises orconsists essentially of polyvinyl chloride (PVC), polymethylmethacrylate (PMMA) and decyl methacrylate or copolymers or anycombination thereof.

In certain embodiments, the polymer comprises a biocompatible polymer,e.g., selected from poly(caprolactone) (PCL), ethylene vinyl acetatepolymer (EVA), poly(ethylene glycol) (PEG), poly(vinyl acetate) (PVA),poly(lactic acid) (PLA), poly(glycolic acid) (PGA),poly(lactic-co-glycolic acid) (PLGA), polyalkyl cyanoacrylate,polyethylenimine,dioleyltrimethyammoniumpropane/dioleyl-sn-glycerolphosphoethanolamine,polysebacic anhydrides, polyurethane, nylons, or copolymers thereof. Inpolymers including lactic acid monomers, the lactic acid may be D-, L-,or any mixture of D- and L-isomers. The terms “biocompatible polymer”and “biocompatibility” when used in relation to polymers areart-recognized. For example, biocompatible polymers include polymersthat are neither themselves toxic to the host (e.g., a cell, an animal,or a human), nor degrade (if the polymer degrades) at a rate thatproduces monomeric or oligomeric subunits or other byproducts at toxicconcentrations in the host. Consequently, in certain embodiments,toxicology of a biodegradable polymer intended for intracellular or invivo use, such as implantation or injection into a patient, may bedetermined after one or more toxicity analyses. It is not necessary thatany subject composition have a purity of 100% to be deemedbiocompatible. Hence, a subject composition may comprise 99%, 98%, 97%,96%, 95%, 90% 85%, 80%, 75% or even less of biocompatible polymers,e.g., including polymers and other materials and excipients describedherein, and still be biocompatible.

The polymer phase may comprise a plasticizer, such as dioctyl sebacate(DOS), o-nitrophenyl-octylether, dimethyl phthalate,dioctylphenyl-phosphonate, dibutyl phthalate, hexamethylphosphoramide,dibutyl adipate, dioctyl phthalate, diundecyl phthalate, dioctyladipate, dioctyl sebacate, or other suitable plasticizers. In certainembodiments, the plasticizer is poly(glycerol sebacate), PGS.

In certain embodiments, e.g., particularly where the polymer isbiocompatible, a biocompatible plasticizer is used. The term“biocompatible plasticizer” is art-recognized, and includes materialswhich are soluble or dispersible in the relevant polymer, which increasethe flexibility of the polymer matrix, and which, in the amountsemployed, are biocompatible. Suitable plasticizers are well known in theart and include those disclosed in U.S. Pat. Nos. 2,784,127 and4,444,933. Specific plasticizers include, by way of example, acetyltri-n-butyl citrate (c. 20 weight percent or less), acetyltrihexylcitrate (c. 20 weight percent or less), butyl benzyl phthalate,dibutylphthalate, dioctylphthalate, n-butyryl tri-n-hexyl citrate,diethylene glycol dibenzoate (c. 20 weight percent or less) and thelike.

The ionophore is a compound, typically an electrically neutral compound,that associates (e.g., forms a complex, chelate, or other non-covalentassociation) with a target ion, and is selective for the target ionrelative to other ions. The ionophore is selected to be lipid-solubleand does not emit light in the visible spectrum in either of itscomplexed and non-complexed states. In certain aspects, the ionophore ofthe mixture included herein is chosen to selectively bind an ionicanalyte, for example, K⁺, Na⁺, Ca²⁺, H⁺, Ba²⁺, Li⁺, Cl⁻, NH₄ ⁺, or NO₃⁻. Potassium ion ionophores include, for example, valinomycin, crownethers, e.g., dimethyldibenzo-30-crown-10, dicyclohexyl-18-crown,dimethyldicyclohexyl-18-crown-6, tetraphenyl borate,tetrakis(chlorophenyl)borate. Sodium ion ionophores include, forexample, methyl monensin,N,N′,N″-triheptyl-N,N′,N″-trimethyl-4,4′,4″-propylidintris-(3-oxabutyramide),N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide,4-octadecanoyloxymethyl-N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide,bis[(12-crown-4)methyl]dodecylmethylmalonate. Exemplary calcium ionionophores include, for example, bis(didecylphosphate),bis(4-octylphenylphosphate),bis(4-(1,1,3,3-tetramethylbutyl)phenylphosphatetetracosamethylcyclododecasiloxane,N,N′-di(11-ethoxycarbonyl)undecyl)-N,N′,4,5-tetramethyl-3,6-dioxaoctanediamide. Barium ion ionophores include, for example, calciumdi(2-ethylhexyl)phosphate+decan-1-ol, barium complex ofnonylphenoxypoly(ethyleneoxy)ethanol in ortho-nitrodiphenyl ether.Chloride ion ionophores include, for example,{μ-[4,5-dimethyl-3,6-bis(octyloxy)-1,2-phenylene]}bis(trifluoroacetato-O)dimercuri(ETH 9009),{μ-[4,5-dimethyl-3,6-bis(dodecyloxy)-1,2-phenylene]}bis(mercurychloride) (ETH 9033), 5,10,15,20-tetraphenyl-21H,23H-porphin manganese(III) chloride (MnTPPCl), tributyltin chloride (TBTCl) and trioctyltinchloride (TOTCl). Bicarbonate ion ionophores of the invention include,for example, quaternary ammonium ion exchangerp-octodecyloxy-meta-chlorophenyl-hydrazone-mesoxalonitrile. Ammonium ionionophores include, for example, nonactin and monactin. Nitrate ionionophores include, for example, tridodecylhexadecylammoniumnitrate+n-octyl-ortho-nitrophenyl, 1:10 phenanthroline nickel (II)nitrate+para-nitrocymene. Lithium ion ionophores include, for example,N,N′-diheptyl-N,N′,5,5-tetramethyl-3,7-dioxononanediamide),12-crown-4,6,6-dibenzyl-14-crown-4.

A chromoionophore is an ionophore that changes its optical properties inthe visible spectrum depending on the state of complexation.Chromoionophores for use in sensors are typically proton-sensitive dyesthat change absorbance (and fluorescence in many cases) depending on thedegree of protonation, although chromoionophores that change absorbancein response to other ions can also be used. The chromoionophores arepreferably highly lipophilic to inhibit leaching from the sensor matrix.Suitable chromoionophores include Chromoionophore I (i.e.,9-(Diethylamino)-5-(octadecanoylimino)-5H-benzo[a]phenoxazine),Chromoionophore II (i.e.,9-Dimethylamino-5-[4-(16-butyl-2,14-dioxo-3,15-dioxaeicosyl)phenylimino]benzo[a]phenoxazine)and Chromoionophore III (i.e.,9-(Diethylamino)-5-[(2-octyldecyl)imino]benzo[a]phenoxazine).Chromoionophore II exhibits light absorbance peaks at 520 nm and 660 nmand a fluorescent emission peak at 660 nm. Chromoionophore III has lightabsorbance peaks at 500 nm and 650 nm and fluorescent emission peaks at570 nm and 670 nm.

Quantum dots are fluorescent semiconductor nanocrystals having acharacteristic spectral emission, which is tunable to a desired energyby selection of the particle size, size distribution and composition ofthe semiconductor nanocrystal. The emission spectra of a population ofquantum dots have linewidths as narrow as 25-30 nm, depending on thesize distribution heterogeneity of the sample population, and lineshapesthat are symmetric, gaussian or nearly gaussian with an absence of atailing region. Advantageously, the range of excitation wavelengths ofthe quantum dots is broad. Consequently, this allows the simultaneousexcitation of varying populations of quantum dots in a system havingdistinct emission spectra with a single light source, e.g., in theultraviolet or blue region of the spectrum.

In certain embodiments, quantum dots of the sensor described herein are,for example, inorganic crystallites between 1 nm and about 1000 nm indiameter, preferably between about 2 nm and about 50 nm, more preferablyabout 5 nm to 20 nm, such as about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nm. Such quantum dots include a “core” of one ormore first semiconductor materials, and which may be surrounded by a“shell” of a second semiconductor material. A semiconductor nanocrystalcore surrounded by a semiconductor shell is referred to as a“core/shell” semiconductor nanocrystal. The surrounded “shell” will mostpreferably have a bandgap greater than the bandgap of the core materialand can be chosen so to have an atomic spacing close to that of the“core” substrate. The core and/or the shell material can be asemiconductor material including, but not limited to, those of the groupII-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe and thelike) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP,AlSb, AlS, and the like) and IV (Ge, Si, Pb and the like) materials, andan alloy thereof, or a mixture thereof.

In certain aspects, a sensor comprises exactly one quantum dot. Incertain embodiments, a sensor comprises more than one quantum dot, forexample, 2, 3, 4, or 5 quantum dots. In certain embodiments wherein thesensor comprises more than one quantum dot, the sensor comprises two ormore types of quantum dots, each type having a distinct emissionwavelength, e.g., independently selected from, for example, 490, 520,545, 560, 580, 620, 655 nm. The availability of two distinct wavelengthemissions (e.g., one or more quantum dots of wavelength 545 nm and oneor more quantum dots with emission wavelength of 655 nm) may allowimprovements in recording of changes in ion concentration by using theratio of the two distinct signals. Fluctuations in fluorescence that arecommon to both signals should theoretically cancel in a ratio. Thedetectable fluorescence emission of the quantum dot particles mayfluctuate depending on variables including number of quantum dots,quantum dot location within the cell, photobleaching, and possiblechanges in excitation light intensity, all effects that can occur slowlyand are not related to ion presence or concentration. Therefore, effectsincluding number of quantum dots, quantum dot location within the cell,photobleaching, and possible changes in excitation light intensity, maybe attenuated.

In certain embodiments, the fluorescence signal of the quantum dot maytrigger a detectable event within the cell. For example, fluorescencemay in turn excite a secondary dye or quantum dot in the particle thateasily generates reactive oxygen species (ROS). The ROS would thenattack the cell, effectively stimulating necrosis (cell death), whichmay then be detected either visually or using markers sensitive to celldeath. Alternatively, instead of including a secondary component withinthe particle, another particle may be added to the cell or cell culture.This additional particle may, for example, comprise a photo-degradablepolymer membrane. When the primary sensor fluoresces, the emitted lightwill rupture the secondary particle, releasing its contents. Thecontents may, for example, be a drug that is therapeutic or apoptotic,e.g., triggering another detectable event.

The sensor may comprise an additive, e.g., to embed charge sites withinthe polymer phase and/or to help enforce charge neutrality within thesensor 112. For sensors targeting cations, the additive can be any inertand preferably lipophilic component that has a negative chargeassociated with it. For sensors targeting anions, the additive ispositively charged and preferably lipophilic. The additive allows thepolymer phase to carry a corresponding amount of oppositely chargedparticles while maintaining overall charge neutrality of the sensor. Theconcentration ratio of additive to chromoionophore is preferably 1:1,thereby allowing the chromoionphore to become completely protonated ordeprotonated. One suitable additive for sensors targeting negative ionsis potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTFPB). Thelipophilic, anionic component TFPB molecules are retained by the polymerphase, and the potassium ions are either complexed by the ionophore orexpelled into the sample solution through diffusion. In one particularimplementation, the sensor film is composed of a suspension producedfrom about 60 mg of DOS, 30 mg of PVC, and up to about 5 mg of additive,ionophore, and chromoionophore.

In a sample solution, the sensor continuously extracts or expels, forexample, analyte cations depending on ion activity in the samplesolution. The ion activity of a sample solution can be monitored byobserving the fluorescence of a sensor of the invention in the samplesolution. As depicted in FIG. 2, the sensor may fluoresce in thepresence of a cationic analyte 21, and not in the absence of saidanalyte, Mode A. In such embodiments, the chromoionophore 24, of thesensor absorbs photons 25, of a quantum dot 26, when the cationicanalyte 21 is not bound to the ionophore 22. In such embodiments, thewavelength of photons 25 emitted from the quantum dot 26 when excitedwith a light source such as UV or visible light fall within theabsorbance range, e.g., maximum absorbance range, of the chromoionophore24 bound to a proton 23, such that the fluorescence of the quantum dotis attenuated or completely undetectable from outside of the polymermatrix 20 (Mode A, sensor on the left). As the target ion 21 increasesin concentration in solution, the ions 21 are drawn into the polymermatrix 20 to bind with the ion-selective ionophore 22. To maintaincharge neutrality within the polymer matrix 20, protons 23 dissociatefrom the chromoionophore 24 in the sensor and diffuse out of the polymermatrix 20 into the sample solution, altering the absorbance propertiesof the chromoionophore 24. The deprotonated chromoionophore 24 has ashifted absorbance region such that the photons 25 emitted by thequantum dot 26 are no longer absorbed by the chromoionophore 24 (Mode A,sensor on the right). The sensor then emits a detectable signalindicating the presence of the analyte.

In an alternate embodiment for detecting cationic analytes, FIG. 2, ModeB, the quantum dot 27 of the sensor emits photons 25 that are notabsorbed by the chromoionophore 24 in the absence of the cationicanalyte 21. In certain such embodiments, the chromoionophore 24 absorbsphotons 25 of the quantum dot 27 when the cationic analyte 21 is boundto the ionophore 22. In such embodiments, the wavelength of emittedphotons 25 from the quantum dot 27 when excited with a light source suchas UV or visible light, do not fall within the absorbance range, e.g.,the maximum absorbance range, of the chromoionophore 24 when bound to aproton 23, such that the fluorescence of the quantum dot 27 is emittedfrom the polymer matrix 20 (Mode B, sensor on the left). As the targetion 21 increases in concentration in solution, the ions 21 are drawninto the polymer matrix 20 to bind with the ion-selective ionophore 22.To maintain charge neutrality within the polymer matrix 20 of thesensor, protons 23 dissociate from the chromoionophore 24 of the sensorand diffuse out of the polymer matrix 20 into the sample solution,altering the absorbance properties of the chromoionophore 24. Thedeprotonated chromoionophore 24 has a shifted absorbance region suchthat the photons 25 emitted by the quantum dot 27 are absorbed by thechromoionophore 24 (Mode B, sensor on the right). The sensor signal isattenuated or extinguished indicating the presence of the analyte.

In an embodiment for detecting anionic analytes, depicted in FIG. 3,Mode A, the ionophore 22 of the sensor selectively binds an anionicanalyte 28 or a group of anionic analytes. In certain such embodiments,the sensor comprises a chromoionophore 24 which absorbs photons 25emitted from the quantum dot 26 upon excitation, e.g., by light such asUV or visible, when the ionic analyte 28 is not bound to the ionophore22 of the sensor. In such a state, the wavelengths of the photons 25emitted by the quantum dot 26 are within the absorbance range, e.g., themaximum absorbance range, of the chromoionophore 24 in a deprotonatedstate and the fluorescence detected outside of the polymer matrix 20 isattenuated or undetectable from outside the sensor (FIG. 3, Mode A,sensor on the left). As the target ion 28 increases in concentration inthe sample solution, the anionic analyte 28 is drawn into the polymermatrix 20, binding with the ion-selective ionophore 22. To maintaincharge neutrality within the polymer matrix 20, protons 23 diffuse fromthe sample solution into the polymer matrix 20, protonating thechromoionophores 24 such that the absorbance properties are altered. Theprotonated chromoionophore 24 has a shifted absorbance region such thatthe photons 25 of the quantum dot 26 are not absorbed by thechromoionophore 24 (FIG. 3, Mode A, sensor on the right). The sensoremits a detectable fluorescence signal indicating the presence of theanalyte 28.

In an alternate embodiment for detecting anionic analytes, depicted inFIG. 3, Mode B, the ionophore of the sensor selectively binds an anionicanalyte 28 or a group of anionic analytes. In certain such embodiments,the sensor comprises a chromoionophore 24 which does not absorb photons25 emitted from the quantum dot 26, upon excitation, e.g., by light suchas UV or visible, when the ionic analyte 28 is not bound to theionophore 22 of the sensor. In such a state, the wavelengths of thephotons 25 emitted by the quantum dot 26 are outside of the absorbancerange, e.g., the maximum absorbance range, of the chromoionophore 24 ina deprotonated state and the fluorescence detected outside of thepolymer matrix 20 is attenuated or absent (FIG. 3, Mode B, sensor on theleft). As the target ion 28 increases in concentration in the samplesolution, the anionic analyte 28 is drawn into the polymer matrix 20,binding with the ion-selective ionophore 22. To maintain chargeneutrality in the polymer matrix 20, protons 23 diffuse from the samplesolution into the polymer matrix 20, protonating the chromoionophores 24such that the absorbance properties are altered. The protonatedchromoionophore 24 has a shifted absorbance region such that the photons25 of the quantum dot 26 are not absorbed by the chromoionophore 24(FIG. 3, Mode B, sensor on the right). The sensor signal is attenuatedor extinguished indicating the presence of the analyte 28.

The following is a non-limiting, illustrative list of target ion (21 or28)/ionophore 22 pairings suitable for use in the sensors: potassium/Potassium Ionophore III (i.e., BME-44,2-Dodecyl-2-methyl-1,3-propanediylbis[N-[5′-nitro(benzo-15-crown-5)-4′-yl]carbamate]), sodium/SodiumIonophore IV (i.e., 2,3:11,12-Didecalino-16-crown-5 2,6,13,16,19Pentaoxapentacyclo [18.4.4.4^(7,12).0^(1,20).0^(7,12)] dotriacontane),sodium/Sodium Ionophore V (i.e.,4-Octadecanoyloxymethyl-N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide),sodium/Sodium Ionophore VI (i.e.,Bis[(12-crown-4)methyl]dodecylmethylmalonate Dodecylmethylmalonic acidbis[(12-crown-4)methyl ester]), sodium/ Sodium Ionophore X(4-tert-Butylcalix[4]arene-tetraacetic acid tetraethylester),calcium/Calcium Ionophore III (i.e., Calimycin), and calcium/Calciumionophore IV (i.e., N,N-Dicyclohexyl-N′,N′-dioctadecyl-diglycolicdiamide). For target anions, illustrative target ion/ionophore pairingsinclude chloride/Chloride Ionophore III (i.e.,3,6-Didodecyloxy-4,5-dimethyl-o-phenylene-bis(mercury chloride) andnitrite/Nitrite Ionophore I (i.e., Cyanoaqua-cobyrinic acidheptakis(2-phenylethyl ester)).

In certain embodiments, the sensor further comprises a surface modifier(SM). In certain embodiments, the SM comprises a molecule that promotesthe delivery or localization of the sensor within a cell. SMs of theinvention include molecules with a hydrophilic portion 40 and ahydrophobic portion 42, FIG. 4. In certain embodiments, the hydrophobicportion 42 of the SM anchors the SM to the hydrophobic polymer matrix41. In certain embodiments, the SM is disposed on the surface of thesensor, e.g., covers a portion of the surface or covers the entiresurface. Exemplary hydrophobic portions 42 of the SM include but are notlimited to, lipids and hydrophobic polymers. In certain embodiments, thehydrophilic portion 40 of the SM is disposed on the surface of thesensor. An exemplary hydrophilic portion 40 includes, but is not limitedto, polyethylene glycol (PEG). In certain embodiments, the hydrophilicportion (PEG) is bound to the hydrophobic portion (lipid) through alinker (e.g., phosphate, ceramide).

In certain embodiments, the sensor further comprises a targeting moiety.In certain embodiments, the targeting moiety is bound to the polymermatrix. In certain embodiments, the targeting moiety is bound to the SMon the surface of the polymer matrix. The targeting moiety, whichassists the sensor in localizing to a particular target area, entering atarget cell(s), and/or locating proximal to an ion channel, may beselected on the basis of the particular condition or site to bemonitored. The targeting moiety may further comprise any of a number ofdifferent chemical entities. In one embodiment, the targeting moiety isa small molecule. Molecules which may be suitable for use as targetingmoieties in the present invention include haptens, epitopes, and dsDNAfragments and analogs and derivatives thereof Such moieties bindspecifically to antibodies, fragments or analogs thereof, includingmimetics (for haptens and epitopes), and zinc finger proteins (for dsDNAfragments). Nutrients believed to trigger receptor-mediated endocytosisand therefore useful targeting moieties include biotin, folate,riboflavin, carnitine, inositol, lipoic acid, niacin, pantothenic acid,thiamin, pyridoxal, ascorbic acid, and the lipid soluble vitamins A, D,E and K. Another exemplary type of small molecule targeting moietyincludes steroidal lipids, such as cholesterol, and steroidal hormones,such as estradiol, testosterone, etc.

In another embodiment, the targeting moiety may comprise a protein.Particular types of proteins may be selected based on knowncharacteristics of the target site or target cells. For example, theprobe can be an antibody either monoclonal or polyclonal, where acorresponding antigen is displayed at the target site. In situationswherein a certain receptor is expressed by the target cells, thetargeting moiety may comprise a protein or peptidomimetic ligand capableof binding to that receptor. Proteins ligands of known cell surfacereceptors include low density lipoproteins, transferrin, insulin,fibrinolytic enzymes, anti-HER2, platelet binding proteins such asannexins, and biological response modifiers (including interleukin,interferon, erythropoietin and colony-stimulating factor). A number ofmonoclonal antibodies that bind to a specific type of cell have beendeveloped, including monoclonal antibodies specific for tumor-associatedantigens in humans. Among the many such monoclonal antibodies that maybe used are anti-TAC, or other interleukin-2 receptor antibodies; 9.2.27and NR-ML-05 to the 250 kilodalton human melanoma-associatedproteoglycan; and NR-LU-10 to a pancarcinoma glycoprotein. An antibodyemployed in the present invention may be an intact (whole) molecule, afragment thereof, or a functional equivalent thereof. Examples ofantibody fragments are F(ab′)₂, Fab′, Fab, and Fv fragments, which maybe produced by conventional methods or by genetic or proteinengineering.

Other preferred targeting moieties include sugars (e.g., glucose,fucose, galactose, mannose) that are recognized by target-specificreceptors. For example, instant claimed constructs can be glycosylatedwith mannose residues (e.g., attached as C-glycosides to a freenitrogen) to yield targeted constructs having higher affinity binding totumors expressing mannose receptors (e.g., glioblastomas andgangliocytomas), and bacteria, which are also known to express mannosereceptors (Bertozzi, C R and M D Bednarski Carbohydrate Research 223:243 (1992); J. Am. Chem. Soc. 114: 2242, 5543 (1992)), as well aspotentially other infectious agents. Certain cells, such as malignantcells and blood cells (e.g., A, AB, B, etc.) display particularcarbohydrates, for which a corresponding lectin may serve as a targetingmoiety.

The nanosensors of the invention can be incorporated into cells todetect ionic analytes and determine the presence and/or concentration,e.g., with a fluorescence signal. FIG. 1 is a schematic diagram of acell assay system 100 according to an illustrative embodiment of theinvention. The cell assay system 100 includes a support 102, abiological sample holder 104, an excitation light source 106, a lightsensor 108, and a computing device 111.

The support 102 supports a sensor 112, e.g., a film having a suspensionof sensor matrices as described above, for positioning in the biologicalsample holder 104. In various implementations, the sensor 112 is adheredto the support 102 by deposition in a solution of sensor matricesdissolved or dispersed in a solvent, such as in a polar organic solventlike tetrahydrofuran (THF). In such implementations, the support 102 ispreferably formed from a material resistant to the solvent. Materialsresistant to THF include, without limitation, 304 stainless steel; 316stainless steel; acetal polymer (marketed as DELRIN by E. I. du Pont deNemours and Company); bronze; carbon graphite; carbon steel; ceramicAl₂O₃, a perfluoroelastomer compound, such as CHEMRAZ marketed byGreene, Tweed; epoxy; HOSTELRY Calloy (marketed by Haynes International,Inc.); KALES elastomer (marketed by DuPont Performance Elastomers);polychlorotrifluoroethylene; NYLON (marketed by E. I. du Pont de Nemoursand Company); polyetherether ketone (PEEK); polyphenylene sulfide; andPTFE.

The film of the sensor can be produced in various ways. In oneimplementation, as described above, a predetermined amount of the sensormixture (e.g., the combined polymer phase, ionophore, quantum dots,additive, and chromoionophore) is dissolved in a solvent, such as THF.The solution is then deposited, sprayed, or spun onto a surface. Thesolvent evaporates, leaving the sensor film on the surface.

In another implementation, the film is formed from a deposition ofsensor microspheres. To produce the microspheres, a sensor emulsion isformed by injecting a sensor suspension dissolved in THF (e.g., 16 mLTHF/100 mg PVC) into a pH buffered solution. The sensor suspensionincludes approximately 60 mg of DOS, 30 mg of PVC, and up toapproximately 5 mg of chromoionophore, additive, and ionophore. Theemulsion is then submerged in a sonicating water bath. Typically, 50 μLof the sensor suspension/THF solution is injected into 1,000-1,500 μL ofbuffered solution. The resulting emulsion contains a mixture ofspherical sensor particles ranging in size from 200 nm to 20 pm indiameter. In certain embodiments, the nanosensors range in size fromabout 5 nm to about 300 nm in diameter, such as about 20 nm to about 200nm in diameter, e.g., about 100 nm. In certain embodiments, thenanosensors that comprise only one quantum dot range in size from about5 nm to about 50 nm in diameter, such as about 5 nm to about 25 nm indiameter, e.g., 20 nm. In certain embodiments wherein the particles arenon-spherical, the diameter is measured at the widest dimension of thenanosensor. Particles of larger dimension are, of course, readilyprepared.

Sensor materials as discussed herein can be sized and shaped in anysuitable configuration that can be achieved using the polymer. Forexample, in certain embodiments, the nanosensors are non-spherical, suchas a disk or a cube, or even sculpted or molded into a utilitarian oraesthetic shape. A sensor emulsion can be spun, sprayed, or evaporatedonto any surface to create a porous sensor membrane. In certainembodiments, the sensor film can be of a size suitable for theapplication, such as the coating of a glass slide, the bottoms of wellsof a 96-well plate, or even a beverage dispenser, such as a pitcher,tank, or bottle. Films formed from microspheres tend to expose a greatersurface area of sensor to a given sample, yielding improved performancecharacteristics.

In certain aspects, a film of the sensor particles is deposited on thesurface of a support. In certain embodiments, the support is aninstrument that can be placed in a solution such as a glass rod, astirring bar, a straw, or glass beads. In certain embodiments, thesupport is a container in which the ionic solution to be evaluated canbe contained. In certain embodiments, the surface of the support ispartially coated with the sensor particles while in other embodiments,the support surface is entirely coated with the sensor particles. Incertain embodiments, the sensors are incorporated within the support andthe support is sculpted into a desired shape such as a stir bar, a film,or a bead.

In certain embodiments, the sensors are used to detect ions in water orother aqueous solutions. In certain embodiments, the support depositedwith the sensor particles is used to detect the presence of ions in anaqueous solution. In certain exemplary embodiments, the sensors are usedto detect ions in water, e.g., tap water or ground water, to determinethe levels of toxic ions in solution or to determine the hardness of theaqueous solution. In certain exemplary embodiments, the sensors areadded to manufacturing solutions to measure ions during production of,e.g., the mass production of soda, ion-restoring beverages or otherionic drinks. In certain embodiments, the sensors are used in thelaboratory to monitor the ion content of a reaction mixture or stocksolution.

The biological sample holder 104 holds a biological sample for analysisby the cell assay system 100. The biological sample can include cellsadhered to the walls of the biological sample holder 104, for example,in a monolayer, or cells suspended in a liquid buffer. The biologicalsample holder 104 is preferably transparent, or at least includes atransparent region through which the sensor 112 can be excited andthrough which the results of such excitation can be monitored.

The sensor 112 is illuminated with a light source 106 to excite thequantum dots. The light source can be in the UV or visible portion ofthe electromagnetic spectrum, or the light source may generate a widespectrum light. In one implementation, the light source 106 is coupledto the support 102.

The emissions of the sensor 112 is detected, e.g., the fluorescence ofthe sensor is detected by a light sensor 108. The light sensor 108 mayinclude a charge-coupled device, a fluorometer, a photomultiplier tube,or other suitable device for measuring fluorescence. In oneimplementation, a spectrophotofluorometer is as both the light source106 and the light sensor 108. The light sensor 108 may also be coupledto the support 102.

The support 102 may include an agent introduction port 118. The agentintroduction port 118 can include a pipette or an electro-mechanicaldispenser device, such as a solenoid or electrostatically driven plungeror syringe.

The computing device 111 controls the various components of the cellassay system 100. The computing device 111 may be a single computingdevice or multiple computing devices providing the variousfunctionalities used to control the cell assay system. Thesefunctionalities are provided by an excitation control module 126, anagent introduction module 130, and an analysis module 134. Theexcitation control module 126 controls the light source 108 to emit awider or narrower wavelength range of excitation light. The agentintroduction module 130 controls the introduction of an agent into thebiological sample holder 104 via an agent introduction means 118. Theanalysis module 134 analyzes the output of the light sensor 108, e.g.,before and after an agent is introduced into the biological sampleholder 104 to determine the effect of the agent on the cells in thebiological sample holder 104. The analysis module 134 may also controlthe other modules in the computing device, i.e., the excitation controlmodule 126 and the agent introduction module 130, to coordinate an assayprotocol. The computing device 111 and/or devices may also includevarious user interface components, such as a keyboard, mouse, trackball,printer, and display.

A module may be implemented as a hardware circuit comprising custom VLSIcircuits or gate arrays, off-the-shelf semiconductors such as logicchips, transistors, or other discrete components. A module may also beimplemented in programmable hardware devices such as field programmablegate arrays, programmable array logic, programmable logic devices or thelike.

Modules may also be implemented in software for execution by varioustypes of processors. An identified module of executable code may, forinstance, comprise one or more physical or logical blocks of computerinstructions which may, for instance, be organized as an object,procedure, or function. Nevertheless, the executables of an identifiedmodule need not be physically located together, but may comprisedisparate instructions stored in different locations which, when joinedlogically together, comprise the module and achieve the stated purposefor the module. A module of executable code may be a single instruction,or many instructions, and may even be distributed over several differentcode segments, among different programs, and across several memorydevices.

The various modules are in communication with the various devices theycontrol or from which they obtain data. They may be connected over alocal area network, wirelessly, over a bus, or over typical cables knownin the art of computer interfaces for connecting computing devices withperipherals.

FIGS. 5A-5D depict sensor arrangements suitable for use in variousimplementations of the cell assay system 100 of FIG. 1. FIG. 5A shows afirst sensor arrangement that includes a support 202 and a biologicalsample holder 204. The biological sample holder 204 includes a monolayerof cells 206 adhered to the biological sample holder 204. Alternatively,the biological sample holder 204 holds cells suspended in a buffer. Thesupport 202 and biological sample holder 204 correspond to the support102 and biological sample holder 104 of FIG. 1. A sensor film 210 iscoupled to the distal end of the support 202.

FIG. 5B illustrates an alternative sensor arrangement 250, whichincludes a biological sample holder 254 having therein a monolayer ofcells 256 adhered to the surfaces of the biological sample holder 254,or cells suspended in a buffer. Instead of including a sensor filmadhered to a support, the sensor arrangement 250 relies upon nanosensorparticles 258 introduced into the cells 256 adhered to the biologicalsample holder 254.

The nanosensor particles 258 may be introduced into the cells 256 in anysuitable manner. In one method, the particles 258 are introduced into abuffer liquid deposited in the biological sample holder 254. A voltagesource then generates a voltage sufficiently strong to electroporate thecells 256, thereby allowing the nanosensor particles 258 to enterdirectly into the cells. In another approach, the surfaces of thenanosensor particles 258 are first coated with a substance such as ahydrophilic coating, for example polyethyleneglycol (PEG), which assistthe particles 258 in crossing through cell membranes. The nanosensorparticles 258 contact the cells 256 which bring the particles 258 intotheir interior in vesicles via endocytosis, pinocytosis, phagocytosis,or similar biological processes. In certain embodiments, a substanceapplied to the nanosensor particles 258 breaks down the vesiclemembrane, releasing the nanosensor particles 258 into the cellcytoplasm. In still other approaches, the particles 258 may beintroduced into cells 256 using a glass needle or through ballisticbombardment.

To determine compartmentalization of nanosensors within the cells TEMand fluorescence staining can be used. TEM can be used to determinelocation of the nanosensor in a cell, may provide a good understandingof nanosensor transport in the cell and serve as a validation of theco-localization staining. The second method, co-localization staining,can be used to determine endosomal release.

Dyes suitable for performing co-localization studies include: FM1-43,FM4-64, Fluorescein Transferrin, and Lysotracker Red. FM1-43 is alipophilic dye that readily stains cell membranes. Previous studies haveshown the effectiveness of FM1-43 to stain endosomes. Its fluorescenceemission is typically greatly increased upon incorporation into ahydrophobic environment. FM1-43 will typically stain the plasma membraneof a cell and remain associated with the lipid bilayer as it forms anendosome. Dye that is not taken into the cell and remains on the plasmamembrane can be easily removed by gentle washing. FM4-64 is an analog ofFM1-43 and behaves in a very similar fashion. It is more hydrophobicthen FM1-43 and therefore may be more suitable for endocytosis studies.FM4-64 has been well characterized as an endosomal stain. The longwavelength emission of FM4-64 may be advantageous when using sensors ofdifferent spectral properties similar to the other fluorescent stainsbeing utilized.

In some embodiments, the sensor is attached to the exterior of a cellrather than introduced into the interior. If, for example, the activityof an ion channel is to be studied, the sensor may be attached to thecell surface or placed in close proximity to the cell surface in alocation where ion concentrations are in flux, such as adjacent to anion channel. The sensor may be positioned adjacent to the ion channel ofa cell, for example, by covalently linking one or more antibodies thatselectively bind the ion channel of interest to a sensor particle asdescribed above. The antibody-linked sensor particles may be added to acell suspension to bind to the ion channel. This approach can be used tolink ion-specific sensors to any feature on the exterior of the cellmembrane to which antibodies selectively bind. Alternatively, thesensors may be attached to the cell membrane by other suitable couplingchemistries, e.g., biotin-(strept)avidin complexing or polysaccharidebinding. See the thesis “High Throughput Optical Sensor Arrays for DrugScreening” by Daniel I. Harjes (2006), available from the MassachusettsInstitute of Technology and incorporated herein by reference.

FIG. 5C illustrates a second alternative sensor arrangement 270 thatincludes a support 272 and a biological sample holder 274. A sensor film276 is coupled to the distal end of the support 272. A cell monolayer278 adheres to the surfaces of the biological sample holder 274.Alternatively, cells are suspended in a buffer. In addition, nanosensorparticles 280 are introduced into the cells. In certain embodiments, thequantum dots used in the sensor film 276 differ from the quantum dotsused in the nanosensor particles 280. In particular, the differentquantum dots desirably have distinguishable fluorescence characteristicssuch that an analysis module analyzing the output of a light sensormonitoring the sensor arrangement 270 can differentiate between theoutput of the sensor film 272 and the nanosensor particles 280. As aresult, the analysis module can differentiate between intracellulartarget ion concentration and extracellular target ion concentration. Inan exemplary embodiment, the sensor film comprises quantum dots of aselected fluorescence wavelength, e.g., 560 nm, and the nanosensorparticles comprise quantum dots of a selected fluorescence wavelength,e.g., 655 nm. In addition, the sensor film 272 may include ionophoresdifferent from those included in the sensor particles 280, e.g.,nanosensor particles comprising sodium ionophores and sensor filmscomprising potassium ionophores. Thus, the sensor arrangement 270 canmonitor the concentrations of two different target ions.

FIG. 5D illustrates a third alternative sensor arrangement 290 thatincludes an electrode support 292 and a biological sample holder 294.The biological sample holder 294, in addition to a cell monolayer 296 orcells suspended in a buffer, includes a removable sensor film 298. Theremovable sensor film 298, for example, can be a glass cover slip orother transparent surface coated with a sensor film.

In still another embodiment, the sensor film is coated onto the innersurface of the biological sample holder. And in another approach, toaccommodate multiwell plates, such as the 96-well plate format oftenused in assays, one embodiment of the present invention utilizes roundglass coverslips coated with the sensor film along with the cells to bemonitored. In certain embodiments, larger multiwell plates such as 384-and 1536-well plates are applied with a layer of sensor film disposed ona surface of some or all of the wells. In these embodiments, each wellcontains a single sensor type to track a specific species of interest;the various sensor types may differ in the ionophore employed andutilize quantum dots with fluorescence wavelengths that are the same orsimilar. The compound of interest is then added directly to the well.The multiwell plate is then placed in a fluorometer and the fluorescenceintensity is monitored with time.

In a typical implementation, a plurality of biological sample holdersholding biological samples is provided. Biological samples introducedinto the holders may include cells suspended in a buffer solution, butalternatively cells may be adhered to the walls of the biological sampleholders. Next, sensors are introduced into the biological sample holdersas shown in FIGS. 5A and 5C, and/or are introduced into the cellsthemselves. Alternatively, the sensors can coat the walls of thebiological sample holders. As described above, nanosensor particles canbe introduced either by electroporating the cells via electrodespositioned in the biological sample holders or by the chemistry appliedto the nanosensor particles breaching vesicle membranes within thecells. Similarly, the sensors can be introduced into the cells usingpico-injection, bead loading, a gene gun, or through liposomal deliverytechniques known in the art.

An agent, such as a therapeutic, toxin, biological macromolecule (suchas a nucleic acid, an antibody, a protein or portion thereof, e.g., apeptide), small molecule (of 2000 amu or less, 1000 amu or less or 500amu or less), protein, virus, bacteria, chemical compound, mixture ofchemical compounds, or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian) cells ortissues, or other biologically active agent may be introduced into oneor more of the biological sample holders. In one particularimplementation using an array of biological sample holders, no agent isintroduced into a first row of biological sample holders to preserve acontrol. A first agent is introduced into a second row of biologicalsample holders. Additional agents are added to additional rows of thearray of biological sample holders. The fluorescence of the sensorsintroduced into the biological sample holders may be monitored. Themonitoring preferably begins prior to introduction of the agents andcontinues thereafter. Changes in ion concentration resulting from theintroduced agents are then determined. By comparing the changes in ionconcentration after adding an agent, one can determine the effect of theagent on the cells being tested.

The sensors of the invention can be used to monitor the effects ofpharmaceutical agents on biological systems such as the cardiovascularsystem or the circulatory system. Action potentials generated by cardiacor neural cells in culture are defined by a flux of sodium and potassiuminto and out of the cell. In certain embodiments, the sensors of theinvention measure this ion flux in cardiac cells accurately andspatially in a high throughput manner.

In certain aspects, the sensors are used in the drug discovery process.In certain such embodiments, the sensors are used to measure theefficacy of a therapy. For example, ion-selective sensors may beemployed to monitor the effect of ion channel-modulating drugs. Inalternative embodiments, sensors are used to screen for cytotoxicsubstances by, for example, determining ionic flux in cardiac cells inresponse to a cytotoxic agent and using these values as a comparison fortesting novel therapeutic agents.

In certain aspects, the sensors of the invention are implanted intosmall animals to monitor biological responses to new therapeutic agents.In certain embodiments, the implantable sensors are used to study themechanism of disease in small animals. In certain such embodiments, theanimals, such as rats or mice, are, for example, infected with a diseaseand the biological functions are monitored by detecting the signal ofthe implanted optical sensors. In such embodiments, the animal is placedwithin a monitoring element, e.g., a fluorescent monitoring cell similarto a monitoring element used to take X-rays of small animals, whereinthe quantum dots of the sensors are excited, e.g., with UV light, andfluorescence emitted from the sensors within the animal may be detected.

In various embodiments the invention may be constructed to directlydetect the presence of particular ions. As illustrated in the tablesbelow, it is known in the art that certain diseases affect particularion channels in a cell. Accordingly, assays for those ions utilizing thepresent invention may furnish a diagnostic tool to determine thepresence of particular diseases. Accordingly, the scope of the presentinvention should be understood to also include the application of theheretofore-described subject matter to measure the ions set forth in thefollowing tables, as well as their application to diagnose the presenceof the associated diseases also appearing in the following tables.

Channel-forming Channel Gene unit/ligand OMIM Disease Cation channels:CHRNA1/ACHRA CHRNAI α, ACh 100690 Myasthenia congenita CHRNA4 CHRNA4 α,ACh 118504 Autosomal dominant nocturnal frontal lobe epilepsy CHRNB2CHRNB2 β, ACh 118507 Autosomal dominant nocturnal frontal lobe epilepsyPolycystin-2 PKD2 α 173910 Autosomal dominant polycystic kidney disease(ADPKD) CNGA3 CNGA3 α, cGMP 60053 Achromatopsia 2 (color blindness)CNGB1 CNGB1 β, cGMP 600724 Autosomal recessive retinitis pigmentosaCNGB3 CNGB3 β, cGMP 605080 Achromatopsia 3 Sodium channels: Na.1.1 SCN1Aα 182389 Generalized epilepsy with febrile seizures (GEFS+) Na.1.2 SCN2Aα 182390 Generalized epilepsy with febrile and afebrile seizures) Na.1.4SCN4A α 603967 Paramyotonia congenital, potassium aggressive myotonia,hyperkalemic periodic paralysis Na.1.5 SCN5a α 600163 Long-QT syndrome,progressive familial heart block type 1, Brugada syndrome (idiopathicventricular arrhythmia) SCNIB SCN1B β 600235 Generalized epilepsy withfebrile seizures (GEFS+) ENACα SCNNIA α 600228 Pseudohypoaldosteronismtype 1 (PHA1) ENaCβ SCNN1B β 600760 PHA1, Liddle syndrome (dominanthypertension ENaCγ SCNN1G γ 600761 PHA1, Liddle syndrome Potassiumchannels: K, 1.1. KCNA1 α 176260 Episodic ataxia with myokymia KCNQI/K,LQT1 KCNQ1 α 192500 Autosomal dominant long-QT syndrome (Romano-Ward)Autosomal recessive long-QT syndrome with deafness (Jervell-Lange-Nielsen) KCNQ2 KCNQ2 α 602235 BFNC (epilepsy), also with myokymiaKCNQ3 KCNQ3 α 602232 BFNC (epilepsy) KCNO4 KCNQ4 α 603537 DFNA2(dominant hearing loss) HERG/KCNH2 KCNH2 α 152427 Long-QT syndromeKir1.1/ROMK KCNJ1 α 600359 Bartter syndrome (renal salt loss,hypokalemic alkalosis) Kir2.1/IRK/KCNJ2 KCNJ2 α 600681 Long-QT syndromewith dysmorphic features (Andersen syndrome) Kir6.2/KATATP_(ATP) KCNJ11α 600937 Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) SURISURI β 600509 PHHI KCNE1/Mink/ISK KCNE1 β 176261 Autosomal dominantlong-QT syndrome (Romano-Ward) Autosomal recessive long-QT syndrome withdeafness (Jervell- Lange-Nielson) KCNE2/MiRP1 KCNE2 β 603796 Long-QTsyndrome KCNE3/MiRP2 KCNE3 β 604433 Periodic paralysis Calcium channels:Ca, 1.1 CACNA1S α 114208 Hypokalemic periodic paralysis, malignanthyperthermia Ca, 1.4 CACNA1F α 300110 X-linked congenital stationarynight blindness Ca, 2.1 CACNA1A α 601011 Familial hemiplegic migraine,episodic staxia, spinocerebella ataxia type 6 RyRI RYR1 α 180901Malignant hyperthermia, central core disease RyR2 RYR2 α 180902Catecholaminergic polymorphic ventricular tachycardia, arrhythmogenicright ventricular dysplasia type 2 Chloride channels: CFTR ABCC7 α602421 Cystic fibrosis, congenital bilateral asplasia of vas deferenceCIC-1 CLCN1 α 118425 Autosomal recessive (Becker) or dominant (Thomsenmyotonia CIC-5 CLCN5 α 300008 Dent's disease (X-linked proteinuria andkidney stones) CIC-7 CLCN7 α 602727 Osteopetrosis (recessive ordominant) CIC-Kb CLCNKB α 602023 Bartter syndrome type III Barttin BSNDβ 606412 Bartter syndrome type IV (associated with sensorineuraldeafness) GLRA1 GLRA1 α, glycine 138491 Hyperekplexin (startle desease)GABAα1 GABRA1 α GABA 137160 Juvenile myoclonus epilepsy GABAγ2 GABRG2 γ,GABA 137164 Epilepsy Gap junction channels: Cx26 GJB2 121011 DFNB3(autosomal dominant hearing loss) DFNB1 (autosomal recessive hearingloss) Cx30 GJB4 605425 DFNA3 Cx31 GJB3 603324 DFNA2 Cx32 GJB1 304040CMTX (X-linked Charcot-Mari-Tooth neuropathy) AChR α7 Inflammation ClC7Osteoporosis Ether-a-go-go (eag, Cancer erg, elk) Gardos channel Sicklecell anemia P2X7 Immune disorders TRPC6 Asthma, COPD TRPM1 MelanomaTRPM2 Asthma TRPM4 Immune disorders TRPM7 Stroke TRPM8 Prostate cancerTRPV1 Urinary incontinence, pain The third column classifies channelproteins into α, β, and γ subunits, where α subunits are always directlyinvolved in pore formation, Several β subunits are only accessory (i.e.,do not form pores), as is the case, for example, with SCN1B and barttin.Others (e.g. of ENaC and GABA receptors) participate in pore formation.For ligand-gated channels, the ligand is given. Note that GABA andglycine act from the extracellular side, whereas cGMP is anintracellular messenger. Gene Accession ID Gene Locus Tissue ExpressionSodium Channel Type/Disease SCN1A GDB: 118870 2q24 SCN1, vg type 1,α-subunit (280 KDa) Brain S71446 SCN1B GDB: 127281 19q13.1 Hs.89634, vgtype I β₁ subunit Brain, heart, skeletal U12188-12104 (38 KDa) muscleL16242, L10338 SCN2A1 GDB: 120367 2q23 SCN2A, HBSCI, vg type II, α₁-Brain, peripheral nerve subunit (280(KDa) SCN2A2 CDB: 133727 2q23-24.1HBSCH, vg type II, α₂-subunit vg Brain type II, β₂-subunit (33 KDa)SCN2B GDB: 118871 AF019498 SCN3A GDB: 132151 2q24-31 vg type III,α-subunit (280 kDa) Brain S69887 SCN4A GDB: 125181 17q23.1-25.3 SkM1, vgtype IV α-subunit (260 kDa), Skeletal muscle L04216-L04236 hyperkalemicperiodic paralysis, paramyotonia congentia, potassturn-aggravatedmyotonia SCN4B GDB: 125182 3q21 vg type IV, β-subunit, Heart, fetalskeletal SCN5A GDB: 132152 SkM2, hH1, vg type V, α-subunit, muscle longQ-T syndrome 3 SCN6A GDB: 132153 2q21-23 Hs99945, vg type VI, α-subunitHeart, uterus, fetal and denervated skeletal muscle Calcium ChannelType/Disease SCN7A GDB: 228137 12q13 vg type VII, α-subunit Brain,spinal cord SCN8A GDB: 631695 vg type VIII, α-subunit, motor end-platedisease + ataxia in mice SCN9A GDB: 3750013 vg type IX, α-subunitThyroid and adrenal neuroendocrine type gland SCN10A GDB: 7500141pter-p36.3 hPN3, vg type X Sensory neurons, dorsal root ganglia SCNN1AGDB: 366596 12pt3 SCNN1, nvg type 1 α-subunit of Kidney, lung colonZ92978 ENaC SCNN1B GDB: 434471 16p12.2-p12.1 nvg 1 β-subunit, Liddle'ssyndrome Kidney, lung colon pseudohypoaldosterontsm I SCNN1D GDB:6053678 1p36.3-p36.2 DnaCh, nvg 1 δ-subunit Kidney, lung, colon SCNN1GGDB: 568769 16p122-p12.1 nvg 1 γ-subunit, Liddle's syndrome Kidney,lung, colon X87160 pseudohypoaldosterontsm I CACNA1A GDB: 126432 19p13P/Q type α_(1A-)subunit, eqisodic ataxia Brain (cortex, bulbus, CACNL1A4Z80114-Z80155, 19p13.1 2, familial hemiplegic migraine, olfacorius,X99697, U79666 spinocerebellar ataxia 6; tottering, hippocarnpus,leaner, and rolling mice cerebellum, brain stem), motoneurons, kidneyCACNA1B GDB: 580689 9q34 CACNN, N-type α_(1A-)subunit Central,peripheral CACNL1A5 M94172, M94173 nervous system CACNA1C GDB: 12609412p13 CCHL1A1, L-type α_(1A-)subunit Heart, fibroblasts, lung, CACNL1A1L29636, L29634, 12p13.3 smooth muscle (2 splice L29629 variants) CACNA1DGDB: 128872 3p14.3 CCHL1A2, L-type α_(1D)-subunit Brain, pancreas,CACNL1A2 3p21.3.2? neuroendocrine CACNA1E GDB: 434408 1q25-31 R-typeα_(1C-)subunit Brain, skeletal muscle CACNL1A6 (end plate) CACNA1F GDB:6053864 Xp11.23-11.22 α_(1F)-Subunit Retina GACNIAG AF27964 17q22 T-typeα_(1G)-subunit Brain CACNA1S GDB: 126431 1q31-32 L-type α_(1B)-subunit(5% 212, 95% Skeletal muscle (brain, CACNL1A8 Z22672, L33798 190 kDa),malignant hyperthermia kidney) U30666-U30707 5, hypokalernic periodicparalysis CACNA2 GDB: 132010 7q21-22 CACNA2, CACNA2D1, α₈ ₈ -subunitα_(2A); skeletal muscle, CACNL2A Z28613, Z28609 (175 kDa), MHS3 heart,brain, ileum; α_(2B); Z28605, Z28602 brain; α_(2CVD); aorta Z28699,M76559 CACNB1 GDB: 132012 17q21-22 β₁-Subunit (524 aa, 54 kDa) β₁A/M;skeletal muscle CACNLB1 GDB: 1073281 β₁B/C; brain, heart, U86952-U86961spleen M76560, L06111 GDB: 193328 CACNB2 GDB: 132014 10p12 MYSB,β₂-subunit β₂A/B/E; brain, heart, CACNLB2 Q08289 lung, aorta CACNB3 GDB:341023 12q13 β₂-subunit (482 aa) Brain, heart, lung, spleen, CACNLB3L27584 skeletal and smooth muscle, aorta, trachea, ovary, colon CACNB4GDB: 6028693 2q22-23 β₂-subunit, lethargic mice Brain, kidney CACNG GDB:132015 17q24 γ-Subunit (222 aa, 30 kDa) Skeletal muscle, lung CACNLGL07738 CACNG2 γ2-Subunit, stargazin, absence Brain epilepsy stargazer,waggler mice RYR1 GDB: 120359 19q13.1 Ryanodine receptor 1, Ca releaseSkeletal muscle, testis, channel, 3 splice variants, brain, submaxillaryand malignant hyperthermia 1, central adrenal glands, spleen coredisease RYR2 GDB: 125278 1pter-qter RYR2, calcium release channel Heart,smooth muscle 1q42.1-43 RYR3 GDB: 138451 15q14 RYR3, calcium releasechannel Brain, neonatal skeletal 15q14-15 muscle, adult diaphragmPotassium Channel Type/Disease KCNA1 GDB: 127903 12p13 RBK1, HUK1, MBK1,AEMK, Brain, nerve, heart, LO2750 Kv1.1, Shaker homolog 1, Shaker,skeletal muscle, retina, episodic ataxia 1 (with myokymia) pancreaticislet KCNA1B 3q26.1 Kvβ1.1, Kvβ1.3 (splice product), β-subunit KCNA2GDB: 128062 12pter-qter HK4, Kv1.2, Shaker homolog 2 Brain, nerve,heart, X17622 pancreatic islet KCNA2B 1p36.3 Kvβ1.2, β-subunit KCNA8GDB: 128079 1p13.3 Hs.1750, MK3, HLK3, HPCN3, Skeletal muscle, L23499Kv1.3, Shaker homolog 3 lymphocytes (brain, lung, thymus, spleen) KCNA4GDB: 126730 11p14 Hs.89647, Hs.1854, HK1, HPCN2, Brain, nerve, heart,fetal M60450 Kv1.4, Shaker homolog 4 skeletal muscle, M55514 pancreaticislet KCNA4L GDB: 386059 11q14 Shaker homolog type 4-like KCNA5 GDB:127904 12p13.3-13.2 Hs.89509, HK2, HPCNI, Kv1.5 Brain, heart, kidney,lung, M83254 12p13 Shaker hornolog 5 skeletal muscle, M6045112p13.33-12.31 pancreatic islet KCNA6 GDB: 128080 12p13 HBK2, Kv1.6,Shaker homolog 6 Brain, pancreatic islet X17622 KCNA7 GDB: 12790519q13.3 HAK6, Kv1.7 Shaker homolog 7 KCNA8 see KCNQ1 KCNA9 see KCNQ1KCNA10 GDB: 5885822 Shaker homolog type 10, cGMP activated KCNB1 GDB:128081 20q13.2 Kv2.1, Shab homolog 1 Brain, heart, kidney, retina,skeletal muscle KCNB2 Kv2.2, Shab homolog 2 Brain, heart, retina KCNC1GDB: 128082 11p15.1 Kv3.1, Shaw homolog 1 Brain, skeletal muscle, S56770spleen, lymphocytes M96747 KCNC2 GDB: 127906 19q13.3-13.4 Kv3.2, Shawhomolog 2 Brain KCNC3 GDB: 127907 19q13.3 Kv3.3, Shaw homolog 3 Brain,liver KCNC4 GDB: 127908 1p21 Kv3.4, HKSHIIIC, Shaw homolog 4 Brain,skeletal muscle KCND1 GDB: 128083 Kv4.1, Shal hormolog 1 Brain KCND2GDB: 134771 RK5, Kv4.2, Shal homolog 2 Brain, heart, aorta KCND3 GDB:134772 Kv4.3, KSHIVB, Shal homolog 3 KCNE1 GDB: 127909 21q22.1-22.2MinK, ISK, vg Isk homolog 1 (129 Kidney, submandibular aa), long Q-Tsyndrome 5 gland, uterus, heart, cochlea, retina KCNMA1 GDB: 38603110pter-qter SLO, Hs.62679, α-subunit member Fetal skeletal muscleU09383-4 7q32.1 1, α-subunit of maxiK or BK U02632 channel KCNMB1 GDB:6099615 5q34 hSLO-β, β-subunit member 1 (191 Smooth, fetal skeletalU42600 aa), β-subunit of max1K or BK muscle, brain channel (hippocampus,corpus callosum) KCNN1 U69883 SK(Ca)1, small-conductance Ca- Brain,heart activated K channel, apamin- insensitive KCNN2 SK(Ca)2, apaminsensitive Brain, adrenal gland KCNN3 Y08263 1q? SK(Ca)3,small-conductance Ca- Brain, heart, (human AA285078 activated K channel,intermediate embryonic) skeletal apamin sensitivity muscle, liver KCNN4AF022150 19q13.2 IK1, intermediate-conductance T lymphocytes, colon,AF022797 Ca-activated K channel, KCa4, smooth muscles, AF033021 SK4,Gantos channel prostata, red blood cells, AF000972 neurons KCNQ1 GDB:741244 11p15.5 KCNA9, (KV)LQT1, KQT-like Heart, cochlea, kidney, U40990subfamily member 1, long Q-T lung, placenta, colon syndrome 1 KCNQ2 GDB:9787229, 20q13.3 KQT-like subfamily member 2 (872 Brain Y15065, aa)AF033348 KCNQ3 GDB: 9787230 8q24.22-24.3 KQT-like subfamily member 3(825 Brain AF033347 aa) HERG GDB: 407638 7q35-36 HERG, similar toether-a-go go Brain, heart (eag), Ikr, long Q-T syndrome 2 KCNJ1 GDB:204206 11q24 ROMK1, Kirl.1, Hs.463, Kidney, pancreatic islets U65406,U12541 Bartter/hyperprostaglandin E syndrome KCNJ2 GDB: 27896417pter-qter IRK1, Kir2.1, Hs.1547 Muscle, neural tissue, U12507 heartKCNJ3 GDB: 278325 2q24.1 GIRK1, Kir3.1 Heart, cerebellum U50964 KCNJ4GDB: 374080 22q13.1 HIR, HIRK1, HIRK2, Kir2.3 Heart, skeletal muscle,Z97056 brain KCNJ5 GDB: 547948 11q24 CIR, KATP1, GIRK4, Kir3.4 Heart,pancreas KCNJ6 GDB: 547949 21q22.1 KCNJ7, GIRK2, KATP2, BIR1,Cerebellum, pancreatic U24660 Kir3.2, ataxia, weaver mice islet KCNJ8GDB: 633096 12p11.23] Kir6.1, uKATP, ubiquitious K_(ATP) Brain, heart,skeletal, α-subunit smooth muscle, others KCNJ10 GDB: 3750203 1q22-23]Kir1.2, Kir4.1 Glia KCNJ11 GDB: 7009893 [11p15.1] Kir6.2, BIR, K(ATP)α-subunit, Pancreatic islets hyperinsulinemic hypoglycemia KCNJ12 GDB:4583927 [17p11.1] Kir2.2 KCNJ15 GDB: 6275865 [21q22.2] Kir4.2 KCNJN1GDB: 6108062 [ ] Kir2.2v, subfamily inhibitor 1 SUR1 GDB: 591970[11p15.1] SUR(1), sulfonylurea receptor, Pancreatic islets K(ATP)β-subunit, hyperinsulinemic hypoglycemia SUR2 12p12.1] SUR2, SUR2A, B,sulfonylurea 2A: heart, 2B: brain, liver, receptor 2 (1545-aa),β-subunit of skeletal, smooth muscle, K(ATP) urinary bladder KCNK1 GDB:6045446 1q42-43 DPK, TWIK1 Kidney KCNK2 1q41 TREK1 Brain KCNK3 GDB:9773281 2p23 TASK Kidney

Therapeutic Target Enzyme Family Assay Alzheimer's CMGC ERK2 (P42mapk)Alzheimer's Phospholipase PLA2 Alzheimer's Cyclooxygenases COX2Alzheimer's CaMK MARKI Alzheimer's CaMK MARK2 Alzheimer's AGC PKCalphaAlzheimer's AGC PKCgamma Alzheimer's AGC PKCgamma Alzheimer's Cysteineproteases caspase-3 Alzheimer's Cysteine proteases caspase-6 Alzheimer'sAspartic proteases BACE-1 (beta-secretase) Alzheimer's Asparticproteases cathepsin D Alzheimer's Aspartic proteases cathepsin EAlzheimer's Metalloproteases ACE Alzheimer's Metalloproteases ACEAlzheimer's Metalloproteases TACE Alzheimer's NO synthases constitutiveNOS (cerebellar) Alzheimer's Monoamine acetylcholinesterase&neurotransmitter synthesis & metabolism Alzheimer's Monoamine COMT(catechol-O-methyl &neurotransmitter transferase) synthesis & metabolismAlzheimer's Monoamine MAO-A &neurotransmitter synthesis & metabolismAlzheimer's Monoamine MAO-B &neurotransmitter synthesis & metabolismAlzheimer's Monoamine tyrosine hydroxylase &neurotransmitter synthesis &metabolism Alzheimer's Phospholipase C PLC Alzheimer's Miscellaneousenzymes xanthine oxidase/ superoxide 02-scavenging Dependence/AddictionAGC PKA Dependence/Addiction AGC PKCalpha Dependence/Addiction AGCPKCbeta 1 Dependence/Addiction AGC PKCbeta 2 Dependence/Addiction AGCPKCdelta Dependence/Addiction Monoamine GABA transaminase&neurotransmitter synthesis & metabolism Dependence/Addiction Cyclasesadenylyl cyclase (stimulated) Dependence/Addiction Phospholipase C PLCDependence/Addiction ATPase ATPase (Na⁺/K⁺)Inflammation/Arthritis/Allergy RTK EGFR kinaseInflammation/Arthritis/Allergy RTK FLT-1 kinase (VEGFR1)Inflammation/Arthritis/Allergy RTK KDR kinase (VEGFR2)Inflammation/Arthritis/Allergy CTK Fyn kinaseInflammation/Arthritis/Allergy CTK HCK Inflammation/Arthritis/AllergyCTK Lek kinase Inflammation/Arthritis/Allergy CTK Lyn kinaseInflammation/Arthritis/Allergy CTK ZAP70 kinaseInflammation/Arthritis/Allergy CMGC ERK2 (P42mapk)Inflammation/Arthritis/Allergy CMGC JNK 1 Inflammation/Arthritis/AllergyCMGC JNK 2 Inflammation/Arthritis/Allergy CMGC P38alpha kinaseInflammation/Arthritis/Allergy Phospholipase PLA2Inflammation/Arthritis/Allergy Cyclooxygenases COX1Inflammation/Arthritis/Allergy Cyclooxygenases COX2Inflammation/Arthritis/Allergy TXA2 synthetase TXA2 synthetaseInflammation/Arthritis/ CaMK MAPKAPK2 Allergy Inflammation/Arthritis/AGC PKA Allergy Inflammation/Arthritis/ Lipoxygenases 12-lipoxygenaseAllergy Inflammation/Arthritis/ Lipoxygenases 15-lipoxygenase AllergyInflammation/Arthritis/ Serine proteases elastase AllergyInflammation/Arthritis/ Serine proteases cathepsin G AllergyInflammation/Arthritis/ Serine proteases kallikrein AllergyInflammation/Arthritis/ Serine proteases tryptase AllergyInflammation/Arthritis/ Cysteine proteases caspase-1 AllergyInflammation/Arthritis/ Cysteine proteases caspase-4 AllergyInflammation/Arthritis/ Cysteine proteases caspase-5 AllergyInflammation/Arthritis/ Cysteine proteases cathepsin B AllergyInflammation/Arthritis/ Cysteine proteases cathepsin X AllergyInflammation/Arthritis/ Aspartic proteases cathepsin E AllergyInflammation/Arthritis/ Metalloproteases MMP-1 AllergyInflammation/Arthritis/ Metalloproteases MMP-2 AllergyInflammation/Arthritis/ Metalloproteases MMP-3 AllergyInflammation/Arthritis/ Metalloproteases MMP-7 AllergyInflammation/Arthritis/ Metalloproteases MMP-8 AllergyInflammation/Arthritis/ Metalloproteases MMP-9 AllergyInflammation/Arthritis/ Metalloproteases MMP-13 AllergyInflammation/Arthritis/ Metalloproteases MT1-MMP (MMP-14) AllergyInflammation/Arthritis/ Metalloproteases TACE AllergyInflammation/Arthritis/ Phosphatases phosphatase CD45 AllergyInflammation/Arthritis/ Phosphodiesterases PDE2 AllergyInflammation/Arthritis/ Phosphodiesterases PDE4 AllergyInflammation/Arthritis/ Phosphodiesterases acid sphingomyelinase AllergyInflammation/Arthritis/ Monoamine & HNMT (histamine N- Allergyneurotransmitter methyltransferase) synthesis & metabolismInflammation/Arthritis/ Miscellaneous enzymes myeloperoxidase AllergyInflammation/Arthritis/ Miscellaneous enzymes xanthine oxidase/ Allergysuperoxide 02-scavenging Neuroprotection RTK TRKB Neuroprotection CMGCCDK5 Neuroprotection CMGC DYRKla Neuroprotection CMGC ERK1Neuroprotection CMGC ERK2 (P42mapk) Neuroprotection MCGC JCK 3Inflammation/Arthritis/ Metalloproteases MMP-13 AllergyInflammation/Arthritis/ Metalloproteases MT1-MMP (MMP-14) AllergyInflammation/Arthritis/ Metalloproteases TACE AllergyInflammation/Arthritis/ Phosphatases phosphatase CD45 AllergyInflammation/Arthritis/ Phosphodiesterases PDE2 AllergyInflammation/Arthritis/ Phosphodiesterases PDE4 AllergyInflammation/Arthritis/ Phosphodiesterases acid sphingomyelinase AllergyInflammation/Arthritis/ Monoamine & HNMT (histamine N- Allergyneurotransmitter methyltransferase) synthesis & metabolismInflammation/Arthritis/ Miscellaneous enzymes myeloperoxidase AllergyInflammation/Arthritis/ Miscellaneous enzymes xanthine oxidase/ Allergysuperoxide 02-scavenging Neuroprotection RTK TRKB Neuroprotection CMGCCDK5 Neuroprotection CMGC DYRKla Neuroprotection CMGC ERK1Neuroprotection CMGC ERK2 (P42mapk) Neuroprotection MCGC JCK 3Neuroprotection Cyclooxygenases COXI Neuroprotection CyclooxygenasesCOX2 Neuroprotection CaMK CaMK2alpha Neuroprotection AGC PKANeuroprotection Cysteine proteases caspase-3 NeuroprotectionPhosphodiesterases PDEI Neuroprotection Phosphodiesterases PDE6Neuroprotection NO synthases constitutive NOS (endothelial)Neuroprotection NO synthases constitutive NOS (cerebellar)Neuroprotection Monoamine & acetylcholinesterase neurotransmittersyntheses & metabolism Neuroprotection Monoamine & COMT(catechol-O-methyl neurotransmitter transferase) syntheses & metabolismNeuroprotection Monoamine & GABA transaminase neurotransmitter syntheses& metabolism Neuroprotection Monoamine & HNMT (histamine N-neurotransmitter methyltransferase) syntheses & metabolismNeuroprotection Monoamine & MAO-A neurotransmitter syntheses &metabolism Neuroprotection Monoamine & MAO-A neurotransmitter syntheses& metabolism Neuroprotection Monoamine & PNMT neurotransmitter(phenylethanoiamine-N- syntheses & metabolism methyl transferase)Neuroprotection Monoamine & tyrosine hydroxylase neurotransmittersyntheses & metabolism Neuroprotection Cyclases guanylyl cyclase (basal)Neuroprotection Cyclases guanylyl cyclase (stimulated) NeuroprotectionATPase ATPase (Na+/K+) Neuroprotection Miscellaneous enzymes xanthineoxidase/superoxide 02- scavenging Parkinson CMGC JNK 1 ParkinsonPhospholipase PLA2 Parkinson Cyclooxygenases COX2 Parkinson Cysteineproteases caspase-3 Parkinson NO synthases constitutive NOS (cerebellar)Parkinson Monoamine & acetylcholinesterase neurotransmitter syntheses &metabolism Parkinson Monoamine & COMT (catechol-O-methylneurotransmitter transferase syntheses & metabolism Parkinson Monoamine& MAO-A neurotransmitter syntheses & metabolism Parkinson Monoamine &MAO-B neurotransmitter syntheses & metabolism Cancer RTK Axl kinaseCancer RTK c-kit kinase Cancer RTK c-kit kinase Cancer RTK EGFR kinaseCancer RTK EphAl kinase Cancer RTK EphA3 kinase Cancer RTK EphA4 kinaseCancer RTK EphB2 kinase Cancer RTK FGFR1 kinase Cancer RTK FGFR2 kinaseCancer RTK FGFR3 kinase Cancer RTK FGFR4 kinase Cancer RTK FLT-1 kinase(VEGFRl) Cancer RTK FLT-3 kinase Cancer RTK FLT-4 kinase (VEGFR3) CancerRTK Fms/CSFR kinase Cancer RTK HER2/ErbB2 kinase Cancer RTK HER4/ErbB4kinase Cancer RTK KDR kinase (VEGFR2) Cancer RTK PDGFRalpha kinaseCancer RTK PDGFRbeta kinase Cancer RTK Ret kinase Cancer RTK TIE2 kinaseCancer RTK TRKA Cancer CTK Abl kinase Cancer CTK BLK Cancer CTK BMX (Bk)kinase Cancer CTK BRK Cancer CTK BTK Cancer CTK CSK Cancer CTK FAKCancer CTK Fes kinase Cancer CTK Fyn kinase Cancer CTK JAK2 Cancer CTKJAK3 Cancer CTK Lck kinase Cancer CTK PYK2 Cancer CTK Src kinase CancerCTK Syk Cancer CTK Yes kinase Cancer CMGC CDC2/CDK1 (cycB) Cancer CMGCCDK2 (cycE) Cancer CMGC CDK4 (cycDl) Cancer CMGC CDK5 Cancer CMGC CK2(casein kinase 2) Cancer CMGC DYRKla Cancer CMGC ERK1 Cancer CMGC ERK2(P42mapk) Cancer CMGC HIPK2 Cancer CMGC IKKalpha Cancer CMGC IKKbetaCancer CMGC JNK 1 Cancer CMGC JNK 2 Cancer CMGC NEK1 Cancer CMGC NEK2Cancer CMGC NEK4 Cancer CMGC p38alpha kinase Cancer CMGC p38beta 2kinase (SAPK2b2) Cancer CMGC p38delta kinase Cancer CMGC p38ganunakinase Cancer Cyclooxygenases COX2 Cancer CaMK CaMKldelta Cancer CaMKCaMK Cancer CaMK CHK1 Cancer CaMK CHK2 Cancer CaMK DAPK1 Cancer CaMKDAPK2 Cancer CaMK MAPKAPK2 Cancer CaMK MAPKAPK3 Cancer CaMK MAPKAPK5(PRAKO Cancer CaMK MAARK1 Cancer CaMK MARK2 Cancer CaMK MARK4 CancerCaMK Pim 1 kinase Cancer CaMK Pirn2 kinase Cancer AGC Aktl/PKBalphaCancer AGC Akt2/PKBbeta Cancer AGC Akt3/PKBgamma Cancer AGC AurA/Aur2kinasewhile the red stains the nuclei of dead cells. No difference in theratio of live to dead cells was noted between nanosensor loaded cellsand control (no nanosensors). Following an implementation of thismethod, it was noted that the number of living cells was not differentfrom control cells (not shown) which contained no nanosensors.

Nanosensors in Cardiac Cells: Isolated neonatal rat ventricular myocyteswere plated onto laminin coated 25 mm coverslips. Electroporation wasperformed in a custom chamber with custom parallel electrodes spaced at1 cm. The cells were porated in a Ringer's solution containing 1:10volume ratio of nanosensor solution. 800 V pulses were applied for 20μseconds 8 times using an electroporator (Harvard Apparatus). The cellswere then allowed to recover for 10 minutes before imaging. Images weretaken on a confocal microscope (LSM 510 Meta, Zeiss) exciting at 488 nmand emitting at 650-690, 63× oil immersion objective. As can be seen inFIG. 13 a, the nanosensors loaded into the cardiac cells efficientlyusing electroporation.

As the cells “beat” during electrical stimulation in cardiac cells, thefluorescence from the sodium sensors was collected and was seen tooscillate at 1 Hz, the pacing frequency. In FIG. 13 b, the fluorescencecollected from a nanosensor during stimulation, is charted. The changesof fluorescence observed are attributed to oscillation of sodium, as thechanges occur at the pacing frequency of the cells. The data is theaverage of three time segments of the same experiment (data from time0-3 seconds was averaged with data 3-6 seconds and data 6-9 seconds),and base-line corrected to account for photobleaching.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Therapeutic Target Enzyme Family Assay Cancer AGC AurB/Aur1 kinaseCancer AGC AurC/Aur3 kinase Cancer AGC P70S6Ke Cancer AGC PDK1 CancerAGC PKA Cancer AGC PKCalpha Cancer AGC PKCbeta 1 Cancer AGC PKCbeta 2Cancer AGC PKCdelta Cancer AGC PKCgamma Cancer AGC PKG2 Cancer AGC ROCK1Cancer AGC ROCK2 Cancer AGC RSK2 Cancer AGC SGKI Cancer. Lipoxygenases12-lipoxygenase Cancer TKL RAF-1 kinase Cancer STE MEK1/MAP2KI CancerSTE MKK4/JNK1 Cancer STE MKK6 Cancer STE PAK1 Cancer STE PAK2 CancerSerine proteases elastase Cancer Serine proteases cathepsin G CancerCysteine proteases caspase-2 Cancer Cysteine proteases caspase-3 CancerCysteine proteases caspase-8 Cancer Cysteine proteases caspase-9 CancerCysteine proteases cathepin B Cancer Cysteine proteases cathepsin HCancer Cysteine proteases cathepsin L Cancer Cysteine proteasescathepsin X Cancer Aspartic proteases cathepsin D Cancer Asparticproteases cathepsin E Cancer Metalloproteases MMP-1 CancerMetalloproteases MMP-2 Cancer Metalloproteases MMP-3 CancerMetalloproteases MMP-7 Cancer Metalloproteases MMP-8 CancerMetalloproteases MMP-9 Cancer Metalloproteases MMP-12 CancerMetalloproteases MMP-13 Cancer Metalloproteases MT1-MMP (MMP-14) CancerMetalloproteases TACE Cancer′ Metalloproteases MMP-1 Cancer PhosphatasesPhosphatase 1B Cancer Phosphatases Phosphatase 2B CancerPhosphodiesterases PDE2 Cancer Phosphodiesterases PDE4 CancerPhosphodiesterases PDES Cancer Phosphodiesterases acid spingomyelinaseCancer NO synthases constitutive NOS (endothelial) Cancer NO synthasesconstitutive NOS (cerebellar) Cancer Cyclases adenylyl cyclase (basal)Cancer Cyclases adenylyl cyclase (stimulated) Cancer Phospholipase C PLCCancer Miscellaneous enzymes myeloperoxidase Cancer Miscellaneousenzymes xanthine oxidase/superoxide 02- scavenging Diabetes RTK Ax1kinase Diabetes RTK EGFR kinase Diabetes RTK IGFIR kinase Diabetes CMGCERK2 (P42mapk) Diabetes CMGC Jnk1 Diabetes Cyclooxygenases COX2 DiabetesTXA2 synthetase TXA2 synthetase Diabetes CaMK AMPKalpha Diabetes AGCAkt1/PKBalpha Diabetes AGC Akt2/PKBbeta Diabetes AGC Akt3/PKBgammaDiabetes AGC PDK1 Diabetes AGC PKA Diabetes AGC PKCalpha Diabetes AGCPKCbeta I Diabetes AGC PKCbeta 2 Diabetes AGC PKCgamma Diabetes AGC SGK2Diabetes Metalloproteases ACE Diabetes Metalloproteases MMP-1 DiabetesMetalloproteases MMP-2 Diabetes Metalloproteases MMP-3 DiabetesMetalloproteases MMP-7 Diabetes Metalloproteases MMP-8 DiabetesMetalloproteases MMP-9 Diabetes Metalloproteases MT1-MMP (MMP-14)Diabetes Metalloproteases TACE Diabetes Phosphodiesterases PDE3 DiabetesPhosphodiesterases PDE4 Diabetes Phosphodiesterases PDE5 Diabetes NOsynthases constitutive NOS (endothelial) Diabetes Monoamine &acetylcholinesterase neurotransmitter synthesis & metabolism DiabetesMonoamine & GABA transaminase neurotransmitter synthesis & metabolismDiabetes Monoamine & MAO-B neurotransmitter synthesis & metabolismDiabetes Cyclases adenylyl cyclase (basal) Diabetes Miscellaneousenzymes acetylCoA synthetase Diabetes Miscellaneous enzymes HMG-CoAreductase Diabetes Miscellaneous enzymes xanthine oxidase/superoxide 02-scavenging Metabolic Diseases Cyclooxygenases COX2 Metabolic DiseasesAGC PICA Metabolic Diseases Metalloproteases ACE Metabolic DiseasesPhosphodiesterases PDE3 Metabolic Diseases Phosphodiesterases PDE4Metabolic Diseases NO synthases constitutive NOS (endothelial) MetabolicDiseases Miscellaneous enzymes acetylCoA synthetase Metabolic DiseasesMiscellaneous enzymes HMG-CoA reductase Metabolic Diseases Miscellaneousenzymes xanthine oxidase/superoxide 02- scavenging Obesity CTK PYK2Obesity CMGC JNK1 Obesity CaMK AMPJakoga Obesity AGC PKA ObesityMetalloproteases ACE Obesity Metalloproteases ACE Obesity Phosphatasesphosphatase IB Obesity Phosphodiesterases PDE2 ObesityPhosphodiesterases PDE3 Obesity Monoamine & acetylcholinesteraseneurotransmitter synthesis & metabolism Obesity ATPase ATPase (Na+/K+)Obesity Miscellaneous enzymes HMG-CoA reductase ReproductionPhospholipase PLA2 Reproduction Cyclooxygenases COX1 ReproductionCyclooxygenases COX2 Reproduction Phosphodiesterases PDE5 ReproductionNO synthases constitutive NOS (endothelial) Reproduction Cyclasesguanylyl cyclase (stimulated) Cystic Fibrosis Phospholipase PLA2 CysticFibrosis TXA2 synthetase TXA2 synthetase Cystic Fibrosis AGC PKA CysticFibrosis AGC PKCbeta 1 Cystic Fibrosis AGC PKCbeta 2 Cystic FibrosisSerine proteases elastase Cystic Fibrosis Serine proteases cathepsin GCystic Fibrosis Metalloproteases MMP-2 Cystic FibrosisPhosphodiesterases PDE3 Cystic Fibrosis Phosphodiesterases PDE5 CysticFibrosis Cyclases adenylyl cyclase (stimulated) Cystic FibrosisPhospholipase C PLC Cystic Fibrosis Miscellaneous enzymesmyeloperoxidase Immunosuppression RTK EGFR kinase ProfileImmunosuppression CTK JAK3 Profile Immunosuppression CMGC ERK2 (P42mapk)Profile Immunosuppression Cyclooxygenases COX1 Profile ImmunosuppressionCyclooxygenases COX2 Profile Immunosuppression Serine proteases elastaseProfile Immunosuppression Serine proteases cathepsin G ProfileImmunosuppression Serine proteases tryptase Profile ImmunosuppressionCysteine proteases cathepsin B Profile ImmunosuppressionMetalloproteases ECE-1 Profile Immunosuppression Metalloproteases ECE-1Profile Immunosuppression Metalloproteases MMP-1 ProfileImmunosuppression Metalloproteases MMP-2 Profile ImmunosuppressionMetalloproteases MMP-9 Profile Immunosuppression Phosphatasesphosphatase CD45 Profile Immunosuppression Phosphodiesterases PDE4Profile Immunosuppression Phosphodiesterases acid spingomyelinaseProfile Immunosuppression Cyclases adenylyl cyclase (basal) ProfileImmunosuppression Cyclases adenylyl cyclase Profile (stimulated)Migraine Cyclooxygenases COX2 Migraine NO synthases constitutive NOS(cerebellar) Migraine Monoamine & GABA transaminase neurotransmittersynthesis & metabolism Migraine Cyclases guanylyl cyclase (stimulated)Pain CMGC ERK2 (42mapk) Pain Phospholipase PLA2 Pain CyclooxygenasesCOXI Pain Cyclooxygenases COX2 Pain AGC PICA Pain Serine proteaseselastase Pain Metalloproteases MMP-1 Pain Metalloproteases MMP-2Immunosuppression Serine proteases elastase Profile ImmunosuppressionSerine proteases cathepsin G Profile Immunosuppression Serine proteasestryptase Profile Immunosuppression Cysteine proteases cathepsin BProfile Immunosuppression Metalloproteases ECE-1 ProfileImmunosuppression Metalloproteases ECE-1 Profile ImmunosuppressionMetalloproteases MMP-1 Profile Immunosuppression Metalloproteases MMP-2Profile Immunosuppression Metalloproteases MMP-9 ProfileImmunosuppression Phosphatases Phosphatase CD45 ProfileImmunosuppression Phosphodiesterases PDE4 Profile ImmunosuppressionPhosphodiesterases acid spingomyelinase Profile ImmunosuppressionCyclases adenylyl cyclase (basal) Profile Immunosuppression Cyclasesadenylyl cyclase Profile (stimulated) Migraine Cyclooxygenases COX2Migraine NO synthases constitutive NOS (cerebellar) Migraine Monoamine &GABA transaminase neurotransmitter synthesis & metabolism MigraineCyclases guanylyl cyclase (stimulated) Pain CMGC ERK2 (42mapk) PainPhospholipase PLA2 Pain Cyclooxygenases COXI Pain Cyclooxygenases COX2Pain AGC PICA Pain Serine proteases elastase Pain Metalloproteases MMP-1Pain Metalloproteases MMP-2 Pain Metalloproteases MMP-3 PainMetalloproteases MMP-7 Pain Phosphodiesterases PDE4 Pain NO synthasesconstitutive NOS (endothelial) Pain NO synthases constitutive NOS(cerebellar) Pain Monoamine & GABA transaminase neurotransmittersynthesis & metabolism Pain Monoamine & MAO-A neurotransmitter synthesis& metabolism Pain Monoamine & MAO-B neurotransmitter synthesis &metabolism Pain Monoamine & tyrosine hydroxylase neurotransmittersynthesis & metabolism Pain Miscellaneous enzymes xanthineoxidase/superoxide 02- scavenging

FIGS. 6 a-b present another embodiment 300 of a sensor in accord withthe present invention. The sensor comprises a well 310 andmicro-channels, indicated generally at 315, branching successivelytherefrom. The cells are placed in the well 310, sensors with differentspecificities line each of the channels 315, and a solution is flowedfrom the well 310 through the channels 315. When the fluorescence fromthe channels 315 is imaged, the pattern formed by changes in intensitymay be visualized as an optical barcode, as depicted in FIG. 6 b,indicating the presence or absence of the various sensed materials inthe solution.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

EXAMPLES

-   Ion-Selective Polymer Solution. The ion-selective polymer solution    was made from the following components: 30 mg High Molecular Weight    Polyvinyl Chloride, 60 mg Bis-2-Sebacate, 0.1 mg Sodium Ionophore X,    0.1 mg Sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, and    0.1 mg Chromoionophore I. The combined reagents were stirred in 500    μL of tetrahydrofuran (THF) to afford a homogenous solution.-   Ion-Selective Sensor Fabrication. Quantum dots (ITK organic 655,    Invitrogen) were flocculated in a methanol/isopropanol mixture with    the addition of toluene in a 1:1 (v:v) ratio of toluene:quantum dot    solution. The supernatant was removed and the quantum dots were    resuspended in THF containing 3.3 mM 1-decanethiol. To the quantum    dot solution (0.2 nMoles) was added the ion-selective polymer    solution (17.2 nMoles Chromoionophore I, 50 μl) and the mixture was    stirred.    -   Immobilized Polymer Matrix of Sensors. To form an immobilized        polymer matrix of sensors, 1 μl of the polymer/quantum dot        mixture was dropped onto a 5 mm glass coverslip to afford a thin        homogenous matrix. Individual coverslips could then be placed in        96-well plates and experiments carried out.    -   Particle Sensors. To form particle sensors, dichloromethane was        added to the polymer/quantum dot mixture in a 1:1 (v:v) ratio.        This solution was then added directly to 5 mL of H₂O containing        200 μg of dissolved        1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-550]        (PEG-lipid) by injecting through a pipette tip during        sonication. Sonication was performed with a probe-tip sonicator        (Branson) at 15% amplitude during and after addition for a total        of 3 minutes, resulting in plasticized polymer nanosphere        formation. The solution was then passed through a 0.2 μm syringe        filter (Acrodisc, Gelman Laboratory) to remove large particles.-   Particle Sizing and Zeta Potential. Particle size and zeta potential    of the sensors were determined using a nanosizer (Nano Series ZS90,    Malvern).-   Calibration and Selectivity. Selectivity was determined using    immobilized polymer matrix sensors. Parallel measurements were taken    during addition of increasing ion concentration in HEPES (10 mM)    buffered solution (pH=7.4) for response to sodium and potassium (n=4    each). Response was determined by expressing the data as    α=(I−I_(min))/(I_(max)−I_(min)). I is the intensity at the given ion    concentration, I_(min) is the intensity at the minimum signal (0    Na⁺), and I_(max) is the intensity at the maximum signal (1 M Na⁺).    Data were acquired in a Spectramax Gemini EM microplate fluorometer    (Molecular Devices) exciting at 405 nm and emitting at 655 nm. The    immobilized polymer matrix sensors were pretreated with HEPES    buffered solution at pH=5.0, the pH of the sensors in solution. The    baseline was then established in HEPES buffered solution at pH=7.4.    Sodium and potassium solutions in the range of 1 mM to 1 M were    added and the sensor was allowed to equilibrate before measurements    were made. Response was determined by fitting a sigmoidal curve to    the plot of a vs. ion concentration using Origin software, FIG. 7.

In order to prevent dilution effects, the polymer matrix with nobiocompatible coating was immobilized to a glass coverslip forcalibration and selectivity measurements. The dynamic range of thesensor was found to be 1 mM to 1 M, shown spectrally in FIG. 8 a. Byadjusting the ratio of components, the concentration range was tuned tomaximize the resolution at typical levels of intracellular sodium. Inthis case, the resolution was 80 μM at 17 mM, the center of the dynamicrange. This means that a change in fluorescence intensity of 1% wouldcorrespond to a change in concentration of 80 μM, as measured on afluorescence plate reader.

-   Ratiometric Measurements Immobilized polymer matrix sensors    containing both 545 nm and 655 nm quantum dots was made in a similar    fashion to the method described above. In this case, 1 nmole of each    colored (i.e., emission wavelength) quantum dot was used, giving a    total of 2 nmoles of quantum dots just as above. The sensors were    calibrated with sodium ions while recording emission at 545 and 655    nm in the fluorometer, using an excitation wavelength of 405 nm. The    ratio of 655/545 was taken, averaged over the data set and plotted    using the graphing software program Origin. A sigmoidal curve was    fit to the data and a half-maximal response was determined (FIG. 8    b).

A control ion-selective polymer matrix was made similarly to polymermatrix described above, however the control polymer matrix did notcontain Chromoionophore I. Immobilized polymer matrix sensors were madeas described above. The response was determined by measuringfluorescence over 30 minutes after addition of 200 mM sodium solution.(This was performed in parallel with standard immobilized polymer matrixsensors to analyze the response time).

-   Spectral Overlap. Absorbance characteristics of the chromoionophore    were obtained by creating sensors without quantum dots. The sensors    were placed in a 96-well plate and absorbance was measured at    wavelengths ranging from 500 nm to 700 nm. This was performed in the    presence of 0, 1, 10, 100, and 1000 mM sodium solution in pH 7.4    Hepes buffer. From these spectral results, quantum dots were    selected that emit fluorescence at a wavelength that coincides with    the chromoionophore absorbance wavelengths. The overlap was    confirmed by creating a sensor without chromoionophore but with    quantum dots at the ratio mentioned above. A fluorescent spectrum    was then measured by exciting at 400 nm and collecting emission from    600 nm to 700 nm in steps of 5 nm. In the absence of the    chromoionophore, the quantum dot sensors fluoresced at an intensity    that was not affected by the presence or absence of sodium ions.

A quantum dot with fluorescence maximum at 655 nm and a chromoionophorein a sensor that absorbs fluorescence at low sodium ion concentrationsis depicted in FIG. 9. The absorbance of the chromoionophore at 655 nm(gray lines, FIG. 9) decreases as sodium concentration increases,resulting in an increase in fluorescent signal directly related toincreasing sodium concentration. Note the preferred overlap of thequantum dot emission (red line, FIG. 9).

-   -   Particle sensors without quantum dots: A polymer cocktail was        formulated using, for example, traditional amounts of the sensor        components (PVC, plasticizer, ionophore, chromoionophore and        ionic additive) in a 1:1 (v:v) THF/dichloromethane solution. A        probe-tip sonicator was used to sonicate an aliquot of 100 μL of        polymer solution in 5 mL of buffer containing 0.1% PEG modified        lipid. The nanosensors were dried by passing the particle        solution through a nitrogen feed airbrush.

-   Surface Chemistry: The surface chemistry of the sensor can be    changed by varying the concentration of the functionalized PEG    incorporated onto the sensor. As above, zeta potential can be used    to analyze the effectiveness of the coating at any given    functionalized PEG concentration. The concentration of the    functionalized PEG may be changed to optimize the properties of the    sensors to their intended use.

-   Incorporation of Surface modifier onto Nanosphere Surface: The PEG    lipid molecule PEG2000 PE    {1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene    glycol)-2000](Ammonium Salt)} (Avanti Polar Lipids, Alabaster Ala.)    can be used to attach functional groups. The amine functional group    is also available through Avanti Polar Lipids, DSPE-PEG(2000)Amine    {1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Amino(Polyethylene    Glycol)2000](Ammonium Salt)}, and requires no alterations.

The functionalized PEG can be dissolved into HEPES Buffered Saline(HeBS) and added to a solution containing sensor. The mixture may bemixed, e.g., with a vortexer for 1-2 minutes, to ensure ampleinteraction time and destabilization of aggregates. The resultingsensor-PEG can be subjected to acidification (decrease of solution pHfrom 7.4-5.5) and mechanical stress (trituration). The zeta potentialcan be measured (zetasizer) before and/or after acidification andmechanical stress to determine the surface concentration of the SM. Thismay correlate to the ability of the functionalized PEG to coat thesurface of the sensor and withstand chemical and mechanical changesassociated with the endocytotic pathway.

-   Biocompatibility. Biocompatibility was determined by incubating the    sensors with HEK 293 cells (ATCC). The cells were trypsonized from    normal culture and pipetted into a clear 96-well plate at a    concentration of 30,000 cells/well. The cells were grown overnight    in 300 μL of media to allow attachment to the plate. An aqueous    solution (20 μL) containing 10¹¹ particle sensors/mL was added to    each well. For control experiments 20 μL of distilled water was    used. Different particles were used to compare to the particle    sensors. Particles used for comparison include: gold nanoparticles    (colloidal gold 100 nm, SPI) and FluoSpheres (20 nm carboxylate    modified microspheres, Invitrogen). Each type of nanosensor was    tested on 8 wells of cells. The nanosensors were incubated with the    cells overnight and the media was changed the following day.

At 24, 48, and 72 hours following washing, an MTT assay(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) wasperformed (In vitro toxicology assay kit, Sigma). The cells wereincubated with MTT for 2 hours. The MTT reduction product formazan wasthen dissolved and the absorbance of each well was read at 570 and 690nm. The 690 nm absorbance served as background and was subtracted fromthe 570 nm value. The data was then averaged and compared to control togenerate FIG. 10.

-   Loading sensors without quantum dots into cells: One method used for    loading the cell involved incubating a solution of sensors in media    with the cells overnight. The following day, the cells were washed    3× with PBS buffer. The cells were then imaged on the confocal    microscope, with excitation 633 nm and emission 670-690 nm    indicating that some of the polymer nanosensor had loaded into the    cell, with plenty in the cytosol, but the image indicated a    distribution in larger bundles clustered around the nucleus, FIG.    11.-   Loading sensors into cells: A method for loading sensors into cells    involves injecting the sensors into the cytosol of the HEK 293 cells    via a 2 MOhm resistance patch pipette. The sensor solution may be    diluted, e.g., one to one with a 2× intracellular solution. Once a    GOhm seal has been achieved, the membrane ruptures and the sensors    diffuse into the cytosol of the cell. No pressure need be applied to    the patch pipette solution; rather the sensors enter the cytoplasm    by simple diffusion. Time course experiments can be performed by    acquiring sensor signal every minute over the course of 30 minutes    to evaluate the rate of diffusion out of the patch pipette and    homogeneity of distribution once inside the cytosol. Patching of    cells is discussed in detail below.-   Ability to Load into Cell: The sensors can be loaded into the test    cell, such as HEK 293 or HL-1 cells. HEK 293 cells are maintained by    standard cell culture and HL-1 cells are maintained similarly with    alterations. Cells may be plated, e.g., onto 25 mm glass cover slips    in 6 well plates at 25% confluence the day before experiments are to    be carried out. When using HEK 293 cells the cover slips may be    coated with poly-L-lysine, while cover slips for HL-1 cell    experiments may be coated with gelatin and fibronectin.

The sensors in a HeBS solution may be added to 2 mL of cell culturemedia (at volumes not exceeding a ratio of 1:10) to replace the media ineach well. The sensors can be incubated with the cells at 37° C. 5% CO₂,e.g., 10 min-24 hrs. Following incubation, the cover slips may be washedwith HeBS containing 10 mM Glucose warmed to 37° C. and transferred to amicroscope chamber. The chamber may be filled with HeBS with glucose andplaced onto the microscope (microscope experiments may be performed onthe LSCM). Data may be acquired using LSCM. Images may be acquired usinga plan-apochromatic 63×1.4 NA oil immersion lens. Excitation/Emissionsettings may be selected according to the type of sensors being tested,e.g., for particle sensors-PEG Ex/Em of 633/670-690 nm. Loading may beevaluated by determining the quantity of sensors in each cell. A lack ofnuclear loading may indicate either intracellular loading or plasmamembrane incorporation.

-   Ability to Locate Into cytosol: The sensors may be analyzed for    their ability to release from endosomes and enter the cytosol. Both    HEK 293 and HL-1 cells may be prepared for microscopy according to    the methods above. Additionally, the sensors may be loaded as    described above, optionally with organelle-specific dyes. LSCM    images may be acquired using the same microscope configuration    described above. Cells may be loaded with sensors as described above    and fixed for analysis of cytosolic location by TEM.-   Imaging of sensors: After loading via patch pipette the sensors for    both simultaneous patch experiments and independent imaging    experiments may be performed. Images can be acquired with a standard    CCD camera (CoolSnap HQ, Roper Scientific) or a high speed camera    (Cooke, for channel experiments) attached to a Zeiss Axiovert 200    microscope. A standard FITC cube (Chroma) may be used when imaging    CoroNa Green. Channel activity may be controlled with the patch    setup and fluorescence detection may be timed to coordinate with    channel opening.-   Intracellular calibration: Calibration experiments can be performed    in HEK 293 cells. The SENSORs can be calibrated in the cytosol after    injection loading with a pipette. The sodium ionophore gramicidin    (10 μM, Sigma) can be used to transport sodium across the membrane    and Strophanthidin (100 μM, Sigma) can be used to deactivate any    Na-K ATPase in the cells. Two solutions can be made that contain 140    mM Sodium (30 mM NaCl and 110 mM sodium gluconate) and zero Sodium    (30 mM KCl and 110 mM potassium gluconate, to maintain ionic    balance). Both solutions may also contain 10 mM HEPES, 10 mM glucose    and, 1 mM EGTA (sigma) and a pH of 7.4. They can be mixed to achieve    the desired concentration of sodium and perfused into the microscope    chamber. Acquisitions of data may be made every 5 seconds and the    sodium concentration may only be switched after a plateau in signal    has been achieved for over two minutes. Selectivity can be    determined by performing a calibration response to potassium. In    this case, however, valinomycin (Sigma) may be used instead of    gramicidin, all other conditions being the same.

Response repeatability can be determined using the conditions in thesodium calibration. The extracellular concentration of sodium can beswitched back and forth from zero sodium to 50 mM sodium every tenminutes over the course of an hour.

-   Intracellular Response to Ion of Interest: Experiments may be    performed to determine responsiveness of the nanosensors in the    cytosol. Cells prepared for microscope experiments and    functionalized nanosensors may be loaded as described above.    Nanosensors may be imaged on a LSCM as described above. A    description of methods suitable to characterize sodium nanosensor    response follows; when using other ion-specific nanosensor,    different ionophores can be employed.

LSCM images may be from nanosensor-loaded cells in HeBS containing zerosodium. A sodium ionophore such as Valinomycin (10 μM) (Sigma) may beadded to equilibrate the concentration of sodium between theextracellular solution and the cytosol. Sodium concentrations may beincreased in a step-wise manner by addition of a high sodium (1 M) stockHeBS. Images may be acquired after the system is allowed to reachequilibrium (˜2-3 min) and intensity of the nanosensors can be measured.The sodium concentration may be raised, e.g., to 1 M, to establish amaximum concentration value of intensity. The data may then becorrelated to the both the minimum intensity and maximum intensity and acalibration curve can be generated.

In a similar fashion, selectivity of the cytosolic nanosensor may bedetermined using interfering ions and their corresponding ionophores.The addition of interfering ions and the ionophore may be added whileperforming the calibration mentioned above. The acquired calibrationcurve may then be compared to that generated from sodium ion alone and aselectivity coefficient can be determined.

Nanosensor-loaded cells may also be subjected to whole-cell patch-clamp.The cells can be exposed to a drug to induce sodium flux across plasmamembrane channels. Channel activity using patch-clamp can be recordedsimultaneously with nanosensor fluorescence. This allows a directcomparison of this method to measure ion flux with the instant methoddiscussed herein.

-   Whole Cell Patching: Recombinant HEK 293 cells expressing Nav1.7 can    be used to analyze sodium detection of intracellular nanosensors.    Standard whole cell voltage clamp protocols may be employed to    assess channel current density, voltage-dependent activation,    inactivation, and the time course of recovery from inactivation.    Ionic currents may be recorded with whole-cell voltage clamp    methods, using an Axopatch-200B amplifier (Axon Instruments).    Borosilicate glass electrodes with tip resistances 1-3 Mohm can be    used. Command pulses may be generated by 12-bit digital-to-analog    converter controlled by pCLAMP6 software (Axon Instruments).    Experiments may be conducted at 36° C. To measure activity of the    voltage-gated sodium channels, currents can be recorded following a    step change of the holding potential from −120 mV to −20 mV test    potential. The external solution may contain (mmol/L): 30 NaCl, 110    CsCl, 1.8 CaCl₂, 2 CdCl₂, 1 MgCl₂, 10 HEPES, 10 glucose, 1 4-AP.    Intracellular solution may contain (mmol/L): 10 NaCl, 130 CsCl, 5    EGTA, 10 HEPES, 10 glucose. Inactivation and activation kinetics can    be analyzed. Nanosensors may be introduced via patch pipette in the    whole cell configuration, and maximum amplitude of the sodium    current can be measured simultaneously with measurement of sodium    flux optical recording from the nanosensors.

The same experiments may be repeated with recombinant K_(V) 1.3 HEK 293cells to demonstrate specificity of the sodium-sensitive nanosensors. Inthis case the cells may be hyperpolarized to below the reversalpotential for potassium to allow for potassium influx into the cell. Tomeasure the potassium currents, the extracellular solution may contain(mmol/L) NaCl 136, KCl 5.4, MgCl₂ 1, CaCl₂ 1, NaH₂PO₄ 0.33, HEPES 5, anddextrose 10 (pH 7.35, NaOH). For delayed rectifier current recording,nifedipine (5 μmol/L), 4-aminopyridine (2 mmol/L), and atropine (200nmol/L) can be added to suppress L-type calcium current (I_(Ca,L)),transient outward current (I_(to)), and 4-aminopyridine-dependentmuscarinic K⁺ currents. Dofetilide (1 μmol/L) can be added for I_(Ks)recording. For I_(to) recording, nifedipine may be replaced by CdCl₂(200 μmol/L). Na⁺ current (I_(Na)) contamination may be avoided bysubstitution of equimolar Tris-HCl for NaCl and use of tetrodotoxin. Asuitable internal solution for K⁺-current recording may contain (mmol/L)K-aspartate 110, KCl 20, MgCl₂ 1, MgATP 5, LiGTP 0.1, HEPES 10,Na-phosphocreatinine 5, and EGTA 5.0 (pH 7.3, KOH).

-   Validation of sensor response with molecular dyes: The same    experiments carried out on sensors may be performed using a    fluorescein detection microscope cube. The cell impermeant CoroNa    Green salt can be loaded through the patch pipette to determine if    patch pipette loading alters the location and response of the dye.-   Characterization of sensor function: Blockade of sodium channel    using lidocaine and STX can reduce sodium flux into the cell,    resulting in decrease in signal amplitude using optical recordings    from the sensors. Recombinant Nav1.7 HEK 293 cells may be subject to    whole cell patch clamp, and the sensors may be introduced via the    patch clamp. Lidocaine or STX may be perfused into the patch chamber    and peak amplitude of the sodium current can be determined both by    patch clamp (using standard voltage-dependent activation protocol)    and optical recording (sensors) before and after infusion of the    agent.-   Live/Dead Assays After Nanosensor Loading: A fluorescence live/dead    assay, consisting of calcein to stain living cells and ethidium    bromide to stain the nuclei of dead cells, was performed in order to    determine the viability of cells loaded with nanosensors, FIG. 12.    The staining procedure included incubation of nanosensor-loaded    cells (overnight loading, as above) in 8 μM of calcein and 8 μM of    ethidium bromide for 20 minutes at 37° C. 5% CO₂, then rinsing 3×.    The cells were then imaged on the confocal microscope. In FIG. 12    the green indicates healthy cells,

1. A sensor comprising one or more quantum dots, a polymer, an ionophoreand a chromoionophore, wherein in an initial state photons emitted fromthe one or more quantum dots are absorbed by the chromoionophore, andwhen the ionophore associates with the ionic analyte, thechromoionophore stops absorbing photons emitted by the quantum dot.
 2. Asensor comprising one or more quantum dots, a polymer, an ionophore anda chromoionophore, wherein in an initial state photons emitted from theone or more quantum dots are not absorbed by the chromoionophore, andwhen the ionophore associates with the ionic analyte, thechromoionophore absorbs photons emitted by the quantum dot.
 3. Anapparatus for measuring a characteristic within a living cell, theapparatus comprising: (a) a sensor of claims 1 or 2; and (b) circuitryfor detecting the photons.
 4. The sensor of claims 1 or 2, wherein theionic analyte is K⁺, Na⁺, Ba²⁺, Li⁺, NH₄ ⁺, or Ca²⁺.
 5. The sensor ofclaims 1 or 2, wherein the ionic analyte is Cl⁻ or NO₃ ⁻.
 6. The sensorof any of claims 1-5, further comprising a surface modifier.
 7. Thesensor of claim 6, wherein the surface modifier comprises a hydrophilicportion and a hydrophobic portion.
 8. The sensor of claim 7, wherein thehydrophilic portion of the surface modifier is polyethylene glycol. 9.The sensor of claim 7, wherein the hydrophobic portion of the surfacemodifier is a lipid.
 10. The sensor of claim 7, wherein the hydrophobicportion of the surface modifier and the hydrophilic portion of thesurface modifier are bound together through a linker.
 11. The sensor ofclaim 10, wherein the linker is a covalent bond, a phosphate or aceramide.
 12. The sensor of claims 1 or 2, wherein the sensor comprisesmultiple quantum dots, chromoionophore and ionophore.
 13. The sensor ofclaims 1 or 2, wherein the sensor is a film.
 14. The sensor of claims 1or 2, wherein the sensor is a nanoparticle.
 15. The sensor of claim 14,wherein the diameter of the nanoparticle is between 5 nm and 300 nm. 16.The sensor of claim 14, wherein the diameter of the nanoparticle isbetween 20 nm and 200 nm.
 17. The sensors of claim 14, wherein thenanosensor comprises only one quantum dot and has a diameter between 5nm and 50 nm.
 18. A method for detecting a characteristic of a livingcell, the method comprising: (a) contacting the cell with at least onesensor, wherein the sensor comprises a polymer, a quantum dot thatfluoresces at a first wavelength, and a chromoionophore that absorbsphotons of the first wavelength in one state and does not absorb photonsof the first wavelength in a second state, wherein the states areindicative of the characteristic of the cell, and (b) exciting thequantum dot with a light source causing the quantum dot to fluoresce,and (c) detecting a signal from the sensor.
 19. The method of claim 18,wherein the characteristic is the presence of an ionic analyte.
 20. Themethod of claim 19, wherein the sensor further comprises an ionophorethat selectively binds the ionic analyte.
 21. The method of claim 18,further comprising stimulating the cell so as to affect thecharacteristic.
 22. The method of claim 21, wherein stimulating includescontacting the cell with at least one of a compound, a pathogen, apharmaceutical compound, or a potential toxin.
 23. The method of claim18, wherein contacting the cell comprises placing at least one sensor inproximity to the cell.
 24. The method of claim 18, wherein contactingthe cell comprises coupling the at least one sensor to the exteriormembrane of the cell.
 25. The method of claim 24, wherein contacting thecell comprises coupling the at least one sensor to the exterior membraneof the cell proximate to an ion channel of the cell.
 26. The method ofclaim 25, wherein the at least one sensor is coupled to the membrane viaan antibody that specifically binds the ion channel.
 27. The method ofclaim 18, wherein the characteristic is the presence of a non-ionicproduct, and wherein the method further comprises ionizing the product.28. The method of claim 20, wherein in one state the chromoionophore isdeprotonated and in the second state the chromoionophore is protonated.29. The method of claim 28, wherein the ionic analyte is cationic andwhen ionic analyte is associated with the ionophore, the chromoionophoreis deprotonated, and when the ionic analyte is not associated with theionophore, the chromoionophore is protonated.
 30. The method of claim28, wherein the ionic analyte is anionic and when the ionic analyte isassociated with the ionophore, the chromoionophore is protonated, andwhen the ionic analyte is not associated with the ionophore, thechromoionophore is deprotonated.
 31. The method of claim 18, wherein thecell is in an animal.
 32. The method of claim 18, wherein thefluorescence has an intensity indicative of the concentration of ionicanalyte.
 33. The method of claim 18, wherein the signal comprises theintensity of the fluorescence of the at least one sensor.
 34. The methodof claim 33, wherein detecting the signal comprises using a fluorometer.35. The method of claim 20, wherein the ionic analyte is K⁺, Na⁺, Ba²⁺,Li⁺, NH₄ ⁺, or Ca²⁺.
 36. The method of claim 20 wherein the ionicanalyte is Cl⁻ or NO₃.
 37. The method of claim 18, wherein contactingthe cell comprises introducing the sensor into the cell.
 38. The methodof claim 37, wherein introducing the sensor into the cell comprisesincubating the cell with the sensor in a medium.
 39. A method formanufacturing sensors comprising: combining an ionophore, achromoionophore, quantum dots and a polymer in an organic solvent toform a suspension, and adding the suspension to an aqueous liquid underconditions that promote the formation of nanoparticles.
 40. A method formanufacturing sensors comprising: combining an ionophore, achromoionophore, quantum dots and a polymer in an organic solvent toform a suspension, placing the suspension on a surface, and removing theorganic solvent.
 41. The method of claim 18, wherein the sensorcomprises multiple quantum dots, chromoionophore and ionophore.
 42. Themethod of claim 41, wherein the sensor is a film.